Hydrazinyl-substituted heteroaryl compounds and methods for producing a conjugate

ABSTRACT

The present disclosure provides conjugate structures and hydrazinyl-substituted heteroaryl compounds used to produce these conjugates. The disclosure also encompasses methods of production of such conjugates and compounds, as well as methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/310,497, filed Mar. 18, 2016, the disclosure of which is incorporatedherein by reference.

INTRODUCTION

The field of protein-small molecule therapeutic conjugates has advancedgreatly, providing a number of clinically beneficial drugs with thepromise of providing more in the years to come. Protein-conjugatetherapeutics can provide several advantages, due to, for example,specificity, multiplicity of functions and relatively low off-targetactivity, resulting in fewer side effects. Chemical modification ofproteins may extend these advantages by rendering them more potent,stable, or multimodal.

A number of standard chemical transformations are commonly used tocreate and manipulate post-translational modifications on proteins.There are a number of methods where one is able to modify the sidechains of certain amino acids selectively. For example, carboxylic acidside chains (aspartate and glutamate) may be targeted by initialactivation with a water-soluble carbodiimide reagent and subsequentreaction with an amine. Similarly, lysine can be targeted through theuse of activated esters or isothiocyanates, and cysteine thiols can betargeted with maleimides and α-halo-carbonyls.

One significant obstacle to the creation of a chemically altered proteintherapeutic or reagent is the production of the protein in abiologically active, homogenous form. Conjugation of a drug ordetectable label to a polypeptide can be difficult to control, resultingin a heterogeneous mixture of conjugates that differ in the number ofdrug molecules attached and in the position of chemical conjugation. Insome instances, it may be desirable to control the site of conjugationand/or the drug or detectable label conjugated to the polypeptide usingthe tools of synthetic organic chemistry to direct the precise andselective formation of chemical bonds on a polypeptide.

SUMMARY

The present disclosure provides conjugate structures andhydrazinyl-substituted heteroaryl compounds used to produce theseconjugates. The disclosure also encompasses methods of production ofsuch conjugates and compounds, as well as methods of using the same.

Aspects of the present disclosure include a conjugate comprising atleast one modified amino acid residue of formula (I):

wherein:

R¹ and R² are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl;

one of R³ and R⁴ is a polypeptide and the other is selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

R⁵ and R⁶ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR⁵ and R⁶ are cyclically linked to form a 5 or 6-membered heterocyclyl;

Z¹ is selected from CR⁷, N, O and S;

Z² is C or N;

R⁷ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

L is a linker; and

W is a drug or a detectable label.

In some embodiments, Z¹ and Z² are each N.

In some embodiments, Z¹ is CR⁷ and Z² is N.

In some embodiments, Z¹ is O and Z² is C.

In some embodiments, R³ is the polypeptide.

In some embodiments, R⁴ is the polypeptide.

In some embodiments, R⁵ and R⁶ are each independently selected fromalkyl and substituted alkyl.

In some embodiments, R⁷ is hydrogen.

In some embodiments, the linker is of the formula-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³)_(c)-(T⁴-V⁴)_(d)-(T⁵-V⁵)_(e)—, wherein:

a, b, c, d and e are each independently 0 or 1, where the sum of a, b,c, d and e is 1 to 5;

T¹, T², T³, T⁴ and T⁵ are each independently selected from(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(h)—, 4-amino-piperidinyl (4AP), para-aminobenzyl(PAB), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO),para-amino-benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl(MABC), an acetal group, a disulfide, a hydrazine, a carbohydrate, abeta-lactam, an ester, (AA)_(p)-MABO, (AA)_(p)-MABC, (AA)_(p)-PABO,(AA)_(p)-PABC, MABO-(AA)_(p), MABC-(AA)_(p), PABO-(AA)_(p),PABC-(AA)_(p), (AA)_(p)-MABO-(AA)_(p), (AA)_(p)-MABC-(AA)_(p),(AA)_(p)-PABO-(AA)_(p), and (AA)_(p)-PABC-(AA)_(p);

V¹, V², V³, V⁴ and V⁵ are each independently selected from the groupconsisting of a covalent bond, —CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—,—C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂— and—P(O)OH—;

each R¹ and R¹³ is independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

w is an integer from 1 to 20;

n is an integer from 1 to 30;

p is an integer from 1 to 20; and

h is an integer from 1 to 12.

In some embodiments:

EDA is an ethylene diamine having the structure:

wherein q is an integer from 1 to 6, r is 0 or 1, and each R¹² isindependently selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or wherein two adjacent R¹²groups are cyclically linked to form a piperazinyl ring;

PEG is a polyethylene glycol or a substituted polyethylene glycol;

AA is an amino acid residue; and

4AP is

wherein each R¹⁴ is independently selected from hydrogen, alkyl,substituted alkyl, polyethylene glycol, substituted polyethylene glycol,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In some embodiments, each R¹¹ is independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In some embodiments, each R¹² is independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or wherein two adjacent R¹²groups are cyclically linked to form a piperazinyl ring.

In some embodiments, each R¹³ is independently selected from hydrogen,alkyl, substituted alkyl, aryl, and substituted aryl.

In some embodiments, each R¹⁴ is independently selected from hydrogen,alkyl, substituted alkyl, polyethylene glycol, substituted polyethyleneglycol, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl.

In some embodiments, T¹, T², T³, T⁴ and T⁵ and V¹, V², V³, V⁴ and V⁵ areselected from the following table:

T¹ V¹ T² V² T³ V³ T⁴ V⁴ T⁵ V⁵ (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h)—CONR¹¹— (C₁-C₁₂)alkyl —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(C₁-C₁₂)alkyl —CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— — — — —— — (C₁-C₁₂)alkyl —CO— (AA)_(p) — — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—(PEG)_(n) —NR¹¹— — — — — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(PEG)_(n) —NR¹¹— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— — — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (PEG)_(n) —CO— (EDA)_(w) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w)— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — — — — —(C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —SO₂— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (CR¹³OH)_(h) —CO— — — — — — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹— (PEG)_(n) —CO— — — (C₁-C₁₂)alkyl—CONR¹¹— substituted —NR¹¹— (PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl(C₁-C₁₂)alkyl —SO2— (C₁-C₁₂)alkyl —CO— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (C₁-C₁₂)alkyl — (CR¹³OH)_(h) —CONR¹¹— — — — — (C₁-C₁₂)alkyl—CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) —NR¹¹— — — (C₁-C₁₂)alkyl—CO— (AA)_(p) —NR¹¹— (PEG)_(n) —P(O)OH— (AA)_(p) — — — (C₁-C₁₂)alkyl—CO— (EDA)_(w) —CO— (AA)_(p) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO—(CR¹³OH)_(h) —CONR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — (C₁-C₁₂)alkyl—CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — —CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹—(C₁-C₁₂)alkyl —NR¹¹— — —CO— (C₁-C₁₂)alkyl —NR¹¹— — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —(C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— — — — — (C₁-C₁₂)alkyl—CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —CO— MABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— MABC — (AA)_(p) — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— (AA)_(p) - —NR¹¹— (C₁-C₁₂) alkyl —CO— PABC(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) - — — — PABC-(AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) —PABC- — (AA)_(p) (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO —— — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) — PABO —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — (AA)_(p) —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABO — — — — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— PABC — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO—MABC — (AA)_(p) — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —PABC —NR¹¹— — — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p)— PABC — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) —PABC-(AA)_(p) — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p)— — —

In some embodiments, the linker is selected from one of the followingstructures:

wherein:

each f is independently 0 or an integer from 1 to 12;

each n is independently 0 or an integer from 1 to 30;

each y is independently 0 or an integer from 1 to 20;

each h is independently 0 or an integer from 1 to 12;

each p is independently 0 or an integer from 1 to 20;

each R is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

each R′ is independently selected from hydrogen, a sidechain group of anamino acid, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl.

Aspects of the present disclosure include a compound of formula (II):

wherein:

R⁸ is H and R⁹ is —(CR²R⁴)(NR⁶)(NHR⁵), or R⁸ is —(CR¹R³)(NR⁵)(NHR⁶) andR⁹ is H;

R¹, R², R³ and R⁴ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl;

R⁵ and R⁶ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR⁵ and R⁶ are cyclically linked to form a 5 or 6-membered heterocyclyl;

Z¹ is selected from CR⁷, N, O and S;

Z² is C or N;

R⁷ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

L is a linker; and

W is a drug or a detectable label.

In some embodiments, Z¹ and Z² are each N.

In some embodiments, Z¹ is CR⁷ and Z² is N.

In some embodiments, Z¹ is O and Z² is C.

In some embodiments, R⁵ and R⁶ are each independently selected fromalkyl and substituted alkyl.

In some embodiments, R⁷ is hydrogen.

In some embodiments, the linker is of the formula-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³)_(c)-(T⁴-V⁴)_(d)-(T⁵-V⁵)_(e)—, wherein:

a, b, c, d and e are each independently 0 or 1, where the sum of a, b,c, d and e is 1 to 5;

T¹, T², T³, T⁴ and T⁵ are each independently selected from(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (EDA)_(w), (PEG)_(n),(AA)_(p), —(CR¹³OH)_(h)—, 4-amino-piperidinyl (4AP), para-aminobenzyl(PAB), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO),para-amino-benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl(MABC), an acetal group, a disulfide, a hydrazine, a carbohydrate, abeta-lactam, an ester, (AA)_(p)-MABO, (AA)_(p)-MABC, (AA)_(p)-PABO,(AA)_(p)-PABC, MABO-(AA)_(p), MABC-(AA)_(p), PABO-(AA)_(p),PABC-(AA)_(p), (AA)_(p)-MABO-(AA)_(p), (AA)_(p)-MABC-(AA)_(p),(AA)_(p)-PABO-(AA)_(p), and (AA)_(p)-PABC-(AA)_(p);

V¹, V², V³, V⁴ and V⁵ are each independently selected from the groupconsisting of a covalent bond, —CO—, —NR¹¹—, —CONR¹¹—, —NR¹¹CO—,—C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—, —NR¹¹SO₂— and—P(O)OH—;

each R¹¹ and R¹³ are independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

w is an integer from 1 to 20;

n is an integer from 1 to 30;

p is an integer from 1 to 20; and

h is an integer from 1 to 12.

In some embodiments:

EDA is an ethylene diamine having the structure:

wherein q is an integer from 1 to 6, r is 0 or 1, and each R¹² isindependently selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or wherein two adjacent R¹²groups are cyclically linked to form a piperazinyl ring;

PEG is a polyethylene glycol or a substituted polyethylene glycol;

AA is an amino acid residue; and

4AP is

wherein each R¹⁴ is independently selected from hydrogen, alkyl,substituted alkyl, polyethylene glycol, substituted polyethylene glycol,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In some embodiments, each R¹¹ is independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In some embodiments, each R¹² is independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or wherein any two adjacentR¹² groups are cyclically linked to form a piperazinyl ring.

In some embodiments, each R¹³ is independently selected from hydrogen,alkyl, substituted alkyl, aryl, and substituted aryl.

In some embodiments, each R¹⁴ is independently selected from hydrogen,alkyl, substituted alkyl, polyethylene glycol, substituted polyethyleneglycol, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl.

In some embodiments, T¹, T², T³, T⁴ and T⁵ and V¹, V², V³, V⁴ and V⁵ areselected from the following table:

T¹ V¹ T² V² T³ V³ T⁴ V⁴ T⁵ V⁵ (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h)—CONR¹¹— (C₁-C₁₂)alkyl —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(C₁-C₁₂)alkyl —CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— — — — —— — (C₁-C₁₂)alkyl —CO— (AA)_(p) — — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—(PEG)_(n) —NR¹¹— — — — — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(PEG)_(n) —NR¹¹— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— — — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (PEG)_(n) —CO— (EDA)_(w) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w)— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — — — — —(C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —SO₂— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (CR¹³OH)_(h) —CO— — — — — — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹— (PEG)_(n) —CO— — — (C₁-C₁₂)alkyl—CONR¹¹— substituted —NR¹¹  (PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl(C₁-C₁₂)alkyl —SO2— (C₁-C₁₂)alkyl —CO— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (C₁-C₁₂)alkyl — (CR¹³OH)_(h) —CONR¹¹— — — — — (C₁-C₁₂)alkyl—CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) —NR¹¹— — — (C₁-C₁₂)alkyl—CO— (AA)_(p) —NR¹¹— (PEG)_(n) —P(O)OH— (AA)_(p) — — — (C₁-C₁₂)alkyl—CO— (EDA)_(w) —CO— (AA)_(p) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO—(CR¹³OH)_(h) —CONR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — (C₁-C₁₂)alkyl—CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — —CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹—(C₁-C₁₂)alkyl —NR¹¹— — —CO— (C₁-C₁₂)alkyl —NR¹¹— — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —(C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— — — — — (C₁-C₁₂)alkyl—CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —CO— MABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— MABC — (AA)_(p) — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— (AA)_(p) - —NR¹¹— (C₁-C₁₂) alkyl —CO— PABC(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) - — — —PABC-(AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)— PABC-(AA)_(p) — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO— — — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) — PABO —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — (AA)_(p) —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABO — — — — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— PABC — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO—MABC — (AA)_(p) — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —PABC —NR¹¹— — — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p)— PABC — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) —PABC-(AA)_(p) — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p)— — —

In some embodiments, the linker is selected from one of the followingstructures:

wherein

each f is independently 0 or an integer from 1 to 12;

each n is independently 0 or an integer from 1 to 30;

each y is independently 0 or an integer from 1 to 20;

each h is independently 0 or an integer from 1 to 12;

each p is independently 0 or an integer from 1 to 20;

each R is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

each R′ is independently selected from hydrogen, a sidechain group of anamino acid, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl.

Aspects of the present disclosure include a method of producing apolypeptide conjugate, where the method includes combining in a reactionmixture: a compound of the present disclosure, and a polypeptide thatincludes a reactive aldehyde group or a reactive ketone group. Thecombining is under reaction conditions suitable to promote reactionbetween the compound and the reactive aldehyde group or reactive ketonegroup of the polypeptide to form a polypeptide conjugate. The methodfurther includes isolating the polypeptide conjugate from the reactionmixture.

Aspects of the present disclosure include a pharmaceutical composition,which includes a conjugate of the present disclosure, and apharmaceutically acceptable excipient.

Aspects of the present disclosure include a method of delivering aconjugate to a subject. The method includes administering to the subjectan effective amount of a conjugate of the present disclosure.

Aspects of the present disclosure include a method of treating acondition in a subject. The method includes administering to the subjecthaving the condition a therapeutically effective amount of apharmaceutical composition comprising a conjugate of the presentdisclosure, where the administering is effective to treat the conditionin the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a hydrophobic interaction column (HIC) trace of anantibody-drug conjugate produced by conjugating an aldehyde-taggedantibody to a maytansine modified to include a hydrazinyl-substitutedimidazolyl coupling moiety, according to embodiments of the presentdisclosure.

DEFINITIONS

The following terms have the following meanings unless otherwiseindicated. Any undefined terms have their art recognized meanings.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way ofexample, linear and branched hydrocarbyl groups such as methyl (CH₃—),ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl(CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—),t-butyl ((CH₃)₃C—), n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl((CH₃)₃CCH₂—).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain (except the C₁carbon) have been optionally replaced with a heteroatom such as —O—,—N—, —S—, —S(O)_(n)— (where n is 0 to 2), —NR— (where R is hydrogen oralkyl) and having from 1 to 5 substituents selected from the groupconsisting of alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl, —SO₂-heteroaryl, and—NR^(a)R^(b), wherein R′ and R″ may be the same or different and arechosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.

“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

The term “alkane” refers to alkyl group and alkylene group, as definedherein.

The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl”refers to the groups R′NHR″— where R is alkyl group as defined hereinand R″ is alkylene, alkenylene or alkynylene group as defined herein.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkoxyamino” refers to the group —NH-alkoxy, wherein alkoxy isdefined herein.

The term “haloalkoxy” refers to the groups alkyl-O— wherein one or morehydrogen atoms on the alkyl group have been substituted with a halogroup and include, by way of examples, groups such as trifluoromethoxy,and the like.

The term “haloalkyl” refers to a substituted alkyl group as describedabove, wherein one or more hydrogen atoms on the alkyl group have beensubstituted with a halo group. Examples of such groups include, withoutlimitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl,trifluoroethyl and the like.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

“Alkenyl” refers to straight chain or branched hydrocarbyl groups havingfrom 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and havingat least 1 and preferably from 1 to 2 sites of double bond unsaturation.This term includes, by way of example, bi-vinyl, allyl, andbut-3-en-1-yl. Included within this term are the cis and trans isomersor mixtures of these isomers.

The term “substituted alkenyl” refers to an alkenyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO— substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO— substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

“Alkynyloxy” refers to the group —O-alkynyl, wherein alkynyl is asdefined herein. Alkynyloxy includes, by way of example, ethynyloxy,propynyloxy, and the like.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclyl-C(O)—, and substitutedheterocyclyl-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. For example, acylincludes the “acetyl” group CH₃C(O)—

“Acylamino” refers to the groups —NR²⁰C(O)alkyl, —NR²⁰C(O)substitutedalkyl, NR²⁰C(O)cycloalkyl, —NR²⁰C(O)substituted cycloalkyl,—NR²⁰C(O)cycloalkenyl, —NR²⁰C(O)substituted cycloalkenyl,—NR²⁰C(O)alkenyl, —NR²⁰C(O)substituted alkenyl, —NR²⁰C(O)alkynyl,—NR²⁰C(O)substituted alkynyl, —NR²⁰C(O)aryl, —NR²⁰C(O)substituted aryl,—NR²⁰C(O)heteroaryl, —NR²⁰C(O)substituted heteroaryl,—NR²⁰C(O)heterocyclic, and —NR²⁰C(O)substituted heterocyclic, whereinR²⁰ is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonyl” or the term “aminoacyl” refers to the group—C(O)NR²¹R²², wherein R²¹ and R²² independently are selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R²¹ and R²² are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonylamino” refers to the group —NR²¹C(O)NR²²R²³ where R²¹,R²², and R²³ are independently selected from hydrogen, alkyl, aryl orcycloalkyl, or where two R groups are joined to form a heterocyclylgroup.

The term “alkoxycarbonylamino” refers to the group —NRC(O)OR where eachR is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, andheterocyclyl are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclyl-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclyl are as defined herein.

“Aminosulfonyl” refers to the group —SO₂NR²¹R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic and where R²¹ and R²²are optionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group and alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Sulfonylamino” refers to the group —NR²¹SO₂R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R²¹ andR²² are optionally joined together with the atoms bound thereto to forma heterocyclic or substituted heterocyclic group, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 18 carbon atoms having a single ring (such as is present in aphenyl group) or a ring system having multiple condensed rings (examplesof such aromatic ring systems include naphthyl, anthryl and indanyl)which condensed rings may or may not be aromatic, provided that thepoint of attachment is through an atom of an aromatic ring. This termincludes, by way of example, phenyl and naphthyl. Unless otherwiseconstrained by the definition for the aryl substituent, such aryl groupscan optionally be substituted with from 1 to 5 substituents, or from 1to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl,alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.

“Aryloxy” refers to the group —O-aryl, wherein aryl is as definedherein, including, by way of example, phenoxy, naphthoxy, and the like,including optionally substituted aryl groups as also defined herein.

“Amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that atleast one R is not hydrogen.

The term “azido” refers to the group —N₃.

“Carboxyl,” “carboxy” or “carboxylate” refers to —CO₂H or salts thereof.

“Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or“carboxylalkyl” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl, —C(O)O-alkynyl,—C(O)O-substituted alkynyl, —C(O)O-aryl, —C(O)O-substituted aryl,—C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —C(O)O-cycloalkenyl,—C(O)O-substituted cycloalkenyl, —C(O)O-heteroaryl, —C(O)O-substitutedheteroaryl, —C(O)O-heterocyclic, and —C(O)O-substituted heterocyclic,wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“(Carboxyl ester)oxy” or “carbonate” refers to the groups —O—C(O)O—alkyl, —O—C(O)O-substituted alkyl, —O—C(O)O-alkenyl,—O—C(O)O-substituted alkenyl, —O—C(O)O-alkynyl, —O—C(O)O-substitutedalkynyl, —O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl,—O—C(O)O-substituted cycloalkyl, —O—C(O)O-cycloalkenyl, —O—C(O)O—substituted cycloalkenyl, —O—C(O)O-heteroaryl, —O—C(O)O-substitutedheteroaryl, —O—C(O)O-heterocyclic, and —O—C(O)O-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

“Cyano” or “nitrile” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. Examples of suitable cycloalkyl groups include, forinstance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyland the like. Such cycloalkyl groups include, by way of example, singlering structures such as cyclopropyl, cyclobutyl, cyclopentyl,cyclooctyl, and the like, or multiple ring structures such asadamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple rings and having at least onedouble bond and preferably from 1 to 2 double bonds.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10carbon atoms having single or multiple rings and having at least onetriple bond.

“Cycloalkoxy” refers to —O-cycloalkyl.

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms,such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected fromthe group consisting of oxygen, nitrogen, and sulfur within the ring.Such heteroaryl groups can have a single ring (such as, pyridinyl,imidazolyl or furyl) or multiple condensed rings in a ring system (forexample as in groups such as, indolizinyl, quinolinyl, benzofuran,benzimidazolyl or benzothienyl), wherein at least one ring within thering system is aromatic and at least one ring within the ring system isaromatic, provided that the point of attachment is through an atom of anaromatic ring. In certain embodiments, the nitrogen and/or sulfur ringatom(s) of the heteroaryl group are optionally oxidized to provide forthe N-oxide (N→O), sulfinyl, or sulfonyl moieties. The term heteroarylincludes, by way of example, pyridinyl, pyrrolyl, indolyl, thienyl(thiophenyl), imidazolyl, furanyl, oxazolyl, thiazolyl, and the like.Unless otherwise constrained by the definition for the heteroarylsubstituent, such heteroaryl groups can be optionally substituted with 1to 5 substituents, or from 1 to 3 substituents, selected from acyloxy,hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, substituted alkyl, substituted alkoxy, substitutedalkenyl, substituted alkynyl, substituted cycloalkyl, substitutedcycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl,aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro,heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy,oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl, and trihalomethyl.

The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl wherealkylene and heteroaryl are defined herein. This term includes, by wayof example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

“Heteroaryloxy” refers to —O-heteroaryl.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from the group consisting ofnitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or moreof the rings can be cycloalkyl, aryl, or heteroaryl, provided that thepoint of attachment is through the non-aromatic ring. In certainembodiments, the nitrogen and/or sulfur atom(s) of the heterocyclicgroup are optionally oxidized to provide for the N-oxide, —S(O)—, or—SO₂— moieties.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, or from 1 to 3 substituents, selected from alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl, and fused heterocycle.

“Heterocyclyloxy” refers to the group —O-heterocyclyl.

The term “heterocyclylthio” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein.

The term “hydroxyamino” refers to the group —NHOH.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O).

“Sulfonyl” refers to the group SO₂-alkyl, SO₂-substituted alkyl,SO₂-alkenyl, SO₂-substituted alkenyl, SO₂-cycloalkyl, SO₂-substitutedcycloalkyl, SO₂-cycloalkenyl, SO₂-substituted cylcoalkenyl, SO₂-aryl,SO₂-substituted aryl, SO₂-heteroaryl, SO₂-substituted heteroaryl,SO₂-heterocyclic, and SO₂-substituted heterocyclic, wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein. Sulfonyl includes, by way of example, methyl-SO₂—, phenyl-SO₂—,and 4-methylphenyl-SO₂—.

“Sulfonyloxy” refers to the group —OSO₂-alkyl, OSO₂-substituted alkyl,OSO₂-alkenyl, OSO₂-substituted alkenyl, OSO₂-cycloalkyl,OSO₂-substituted cycloalkyl, OSO₂-cycloalkenyl, OSO₂-substitutedcylcoalkenyl, OSO₂-aryl, OSO₂-substituted aryl, OSO₂-heteroaryl,OSO₂-substituted heteroaryl, OSO₂-heterocyclic, and OSO₂ substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

The term “aminocarbonyloxy” refers to the group —OC(O)NRR where each Ris independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thioxo” or the term “thioketo” refers to the atom (═S).

“Alkylthio” or the term “thioalkoxy” refers to the group —S-alkyl,wherein alkyl is as defined herein. In certain embodiments, sulfur maybe oxidized to —S(O)—. The sulfoxide may exist as one or morestereoisomers.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined herein including optionally substituted aryl groupsalso defined herein.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined herein including optionallysubstituted aryl groups as also defined herein.

The term “thioheterocyclooxy” refers to the group heterocyclyl-S—wherein the heterocyclyl group is as defined herein including optionallysubstituted heterocyclyl groups as also defined herein.

In addition to the disclosure herein, the term “substituted,” when usedto modify a specified group or radical, can also mean that one or morehydrogen atoms of the specified group or radical are each, independentlyof one another, replaced with the same or different substituent groupsas defined below.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for substituting for one or more hydrogens(any two hydrogens on a single carbon can be replaced with ═O, ═NR⁷⁰,═N—OR⁷⁰, ═N₂ or ═S) on saturated carbon atoms in the specified group orradical are, unless otherwise specified, —R⁶⁰, halo, ═O, —OR⁷⁰, —SR⁷⁰,—NR⁸⁰R⁸, trihalomethyl, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —SO₂R⁷⁰,—SO₂O⁻ M⁺, —SO₂OR⁷⁰, —OSO₂R⁷⁰, —OSO₂O⁻M⁺, —OSO₂OR⁷⁰, —P(O)(O⁻)₂(M+)₂,—P(O)(OR⁷⁰)O⁻ M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —C(O)OM⁺, —C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰,—OC(S)R⁷⁰, —OC(O)O M⁺, —OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰,—NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰,—NR⁷⁰C(O)NR⁷⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰ isselected from the group consisting of optionally substituted alkyl,cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl, each R⁷⁰ is independentlyhydrogen or R⁶⁰; each R⁸⁰ is independently R⁷⁰ or alternatively, twoR⁸⁰'s, taken together with the nitrogen atom to which they are bonded,form a 5-, 6- or 7-membered heterocycloalkyl which may optionallyinclude from 1 to 4 of the same or different additional heteroatomsselected from the group consisting of O, N and S, of which N may have —Hor C₁-C₃ alkyl substitution; and each M⁺ is a counter ion with a netsingle positive charge. Each M⁺ may independently be, for example, analkali ion, such as K⁺, Na⁺, Li⁺; an ammonium ion, such as ⁺N(R⁶⁰)₄; oran alkaline earth ion, such as [Ca²⁺]_(0.5), [Mg²⁺]_(0.5), or[Ba²⁺]_(0.5) (“subscript 0.5 means that one of the counter ions for suchdivalent alkali earth ions can be an ionized form of a compound of theinvention and the other a typical counter ion such as chloride, or twoionized compounds disclosed herein can serve as counter ions for suchdivalent alkali earth ions, or a doubly ionized compound of theinvention can serve as the counter ion for such divalent alkali earthions). As specific examples, —NR⁸⁰R⁸⁰ is meant to include —NH₂,—NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl andN-morpholinyl.

In addition to the disclosure herein, substituent groups for hydrogenson unsaturated carbon atoms in “substituted” alkene, alkyne, aryl andheteroaryl groups are, unless otherwise specified, —R⁶⁰, halo, —O⁻M⁺,—OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸⁰, trihalomethyl, —CF₃, —CN, —OCN, —SCN,—NO, —NO₂, —N₃, —SO₂R⁷⁰, —SO₃ ⁻M⁺, —SO₃R⁷⁰, —OSO₂R⁷⁰, —OSO₃ ⁻M⁺,—OSO₃R⁷⁰, —PO₃ ⁻²(M⁺)₂, —P(O)(OR⁷⁰)O⁻ M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰,—C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —CO₂ ⁻ M⁺, —CO₂R⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰,—C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰, —OCO₂ ⁻M⁺, —OCO₂R⁷⁰, —OC(S)OR⁷⁰,—NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰,—NR⁷⁰C(O)NR⁷⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰,R⁷⁰, R⁸⁰ and M⁺ are as previously defined, provided that in case ofsubstituted alkene or alkyne, the substituents are not —O⁻M⁺, —OR⁷⁰,—SR⁷⁰, or —S⁻M⁺.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for hydrogens on nitrogen atoms in“substituted” heteroalkyl and cycloheteroalkyl groups are, unlessotherwise specified, —R⁶⁰, —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸,trihalomethyl, —CF₃, —CN, —NO, —NO₂, —S(O)₂R⁷⁰, —S(O)₂O⁻M⁺, —S(O)₂R⁷⁰,—OS(O)₂R⁷⁰, —OS(O)₂O⁻M⁺, —OS(O)₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺,—P(O)(OR⁷⁰)(OR⁷⁰), —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —C(O)OR⁷⁰,—C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰,—OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰C(O)OR⁷⁰,—NR⁷⁰C(S)OR⁷⁰, NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰,where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ are as previously defined.

In addition to the disclosure herein, in a certain embodiment, a groupthat is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3substituents, 1 or 2 substituents, or 1 substituent.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups specifically contemplated herein are limited to substitutedaryl-(substituted aryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (salts withcounterions having acceptable mammalian safety for a given dosageregime). Such salts can be derived from pharmaceutically acceptableinorganic or organic bases and from pharmaceutically acceptableinorganic or organic acids. “Pharmaceutically acceptable salt” refers topharmaceutically acceptable salts of a compound, which salts are derivedfrom a variety of organic and inorganic counter ions well known in theart and include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, formate, tartrate, besylate,mesylate, acetate, maleate, oxalate, and the like.

The term “salt thereof” means a compound formed when a proton of an acidis replaced by a cation, such as a metal cation or an organic cation andthe like. Where applicable, the salt is a pharmaceutically acceptablesalt, although this is not required for salts of intermediate compoundsthat are not intended for administration to a patient. By way ofexample, salts of the present compounds include those wherein thecompound is protonated by an inorganic or organic acid to form a cation,with the conjugate base of the inorganic or organic acid as the anioniccomponent of the salt.

“Solvate” refers to a complex formed by combination of solvent moleculeswith molecules or ions of the solute. The solvent can be an organiccompound, an inorganic compound, or a mixture of both. Some examples ofsolvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.When the solvent is water, the solvate formed is a hydrate.

“Stereoisomer” and “stereoisomers” refer to compounds that have sameatomic connectivity but different atomic arrangement in space.Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers,and diastereomers.

“Tautomer” refers to alternate forms of a molecule that differ only inelectronic bonding of atoms and/or in the position of a proton, such asenol-keto and imine-enamine tautomers, or the tautomeric forms ofheteroaryl groups containing a —N═C(H)—NH— ring atom arrangement, suchas pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Aperson of ordinary skill in the art would recognize that othertautomeric ring atom arrangements are possible.

It will be appreciated that the term “or a salt or solvate orstereoisomer thereof” is intended to include all permutations of salts,solvates and stereoisomers, such as a solvate of a pharmaceuticallyacceptable salt of a stereoisomer of subject compound.

“Pharmaceutically effective amount” and “therapeutically effectiveamount” refer to an amount of a compound sufficient to treat a specifieddisorder or disease or one or more of its symptoms and/or to prevent theoccurrence of the disease or disorder. In reference to tumorigenicproliferative disorders, a pharmaceutically or therapeutically effectiveamount comprises an amount sufficient to, among other things, cause thetumor to shrink or decrease the growth rate of the tumor.

“Patient” or “subject” refers to human and non-human subjects,especially mammalian subjects.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition in a patient, such as amammal (particularly a human) that includes: (a) preventing the diseaseor medical condition from occurring, such as, prophylactic treatment ofa subject; (b) ameliorating the disease or medical condition, such as,eliminating or causing regression of the disease or medical condition ina patient; (c) suppressing the disease or medical condition, for exampleby, slowing or arresting the development of the disease or medicalcondition in a patient; or (d) alleviating a symptom of the disease ormedical condition in a patient.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymeric form of amino acids ofany length. Unless specifically indicated otherwise, “polypeptide,”“peptide,” and “protein” can include genetically coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones. The termincludes fusion proteins, including, but not limited to, fusion proteinswith a heterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, proteins which contain at least oneN-terminal methionine residue (e.g., to facilitate production in arecombinant bacterial host cell); immunologically tagged proteins; andthe like.

“Native amino acid sequence” or “parent amino acid sequence” are usedinterchangeably herein to refer to the amino acid sequence of apolypeptide prior to modification to include a modified amino acidresidue.

The terms “amino acid analog,” “unnatural amino acid,” and the like maybe used interchangeably, and include amino acid-like compounds that aresimilar in structure and/or overall shape to one or more amino acidscommonly found in naturally occurring proteins (e.g., Ala or A, Cys orC, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K,Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S,Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also includenatural amino acids with modified side chains or backbones. Amino acidanalogs also include amino acid analogs with the same stereochemistry asin the naturally occurring D-form, as well as the L-form of amino acidanalogs. In some instances, the amino acid analogs share backbonestructures, and/or the side chain structures of one or more naturalamino acids, with difference(s) being one or more modified groups in themolecule. Such modification may include, but is not limited to,substitution of an atom (such as N) for a related atom (such as S),addition of a group (such as methyl, or hydroxyl, etc.) or an atom (suchas Cl or Br, etc.), deletion of a group, substitution of a covalent bond(single bond for double bond, etc.), or combinations thereof. Forexample, amino acid analogs may include α-hydroxy acids, and α-aminoacids, and the like. Other amino acid analogs may include2-formylglycine (FGly).

The term “carbohydrate” and the like may be used to refer to monomersunits and/or polymers of monosaccharides, disaccharides,oligosaccharides, and polysaccharides. The term sugar may be used torefer to the smaller carbohydrates, such as monosaccharides,disaccharides. The term “carbohydrate derivative” includes compoundswhere one or more functional groups of a carbohydrate of interest aresubstituted (replaced by any convenient substituent), modified(converted to another group using any convenient chemistry) or absent(e.g., eliminated or replaced by H). A variety of carbohydrates andcarbohydrate derivatives are available and may be adapted for use in thesubject compounds and conjugates.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, and multispecific antibodies (e.g., bispecificantibodies), humanized antibodies, single-chain antibodies, chimericantibodies, antibody fragments (e.g., Fab fragments), and the like. Anantibody is capable of binding a target antigen. (Janeway, C., Travers,P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., GarlandPublishing, New York). A target antigen can have one or more bindingsites, also called epitopes, recognized by complementarity determiningregions (CDRs) formed by one or more variable regions of an antibody.

The term “natural antibody” refers to an antibody in which the heavy andlight chains of the antibody have been made and paired by the immunesystem of a multi-cellular organism. Spleen, lymph nodes, bone marrowand serum are examples of tissues that produce natural antibodies. Forexample, the antibodies produced by the antibody producing cellsisolated from a first animal immunized with an antigen are naturalantibodies.

The term “humanized antibody” or “humanized immunoglobulin” refers to anon-human (e.g., mouse or rabbit) antibody containing one or more aminoacids (in a framework region, a constant region or a CDR, for example)that have been substituted with a correspondingly positioned amino acidfrom a human antibody. In general, humanized antibodies produce areduced immune response in a human host, as compared to a non-humanizedversion of the same antibody. Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). In certain embodiments, framework substitutions areidentified by modeling of the interactions of the CDR and frameworkresidues to identify framework residues important for antigen bindingand sequence comparison to identify unusual framework residues atparticular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann etal., Nature 332:323 (1988)). Additional methods for humanizingantibodies contemplated for use in the present invention are describedin U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417;5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO98/45331 and WO 98/45332. In particular embodiments, a subject rabbitantibody may be humanized according to the methods set forth inUS20040086979 and US20050033031. Accordingly, the antibodies describedabove may be humanized using methods that are well known in the art.

The term “chimeric antibodies” refer to antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody may be joined to human constant segments, such asgamma 1 and gamma 3. An example of a therapeutic chimeric antibody is ahybrid protein composed of the variable or antigen-binding domain from amouse antibody and the constant or effector domain from a humanantibody, although domains from other mammalian species may be used.

By “genetically-encodable” as used in reference to an amino acidsequence of polypeptide, peptide or protein means that the amino acidsequence is composed of amino acid residues that are capable ofproduction by transcription and translation of a nucleic acid encodingthe amino acid sequence, where transcription and/or translation mayoccur in a cell or in a cell-free in vitro transcription/translationsystem.

The term “control sequences” refers to DNA sequences that facilitateexpression of an operably linked coding sequence in a particularexpression system, e.g. mammalian cell, bacterial cell, cell-freesynthesis, etc. The control sequences that are suitable for prokaryotesystems, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cell systems mayutilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate the initiation of translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. Linking is accomplished by ligation or throughamplification reactions. Synthetic oligonucleotide adaptors or linkersmay be used for linking sequences in accordance with conventionalpractice.

The term “expression cassette” as used herein refers to a segment ofnucleic acid, usually DNA, that can be inserted into a nucleic acid(e.g., by use of restriction sites compatible with ligation into aconstruct of interest or by homologous recombination into a construct ofinterest or into a host cell genome). In general, the nucleic acidsegment comprises a polynucleotide that encodes a polypeptide ofinterest, and the cassette and restriction sites are designed tofacilitate insertion of the cassette in the proper reading frame fortranscription and translation. Expression cassettes can also compriseelements that facilitate expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment and is at least 60% free,at least 75% free, at least 80% free, at least 85% free, at least 90%free, at least 95% free, at least 98% free, or more than 98% free, fromother components with which it is naturally associated.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

By “reactive partner” is meant a molecule or molecular moiety thatspecifically reacts with another reactive partner to produce a reactionproduct. Exemplary reactive partners include a cysteine or serine of asulfatase motif and Formylglycine Generating Enzyme (FGE), which reactto form a reaction product of a converted aldehyde tag containing aformylglycine (fGly) in lieu of cysteine or serine in the motif. Otherexemplary reactive partners include an aldehyde of an fGly residue of aconverted aldehyde tag (e.g., a reactive aldehyde group) and an“aldehyde-reactive reactive partner”, which comprises analdehyde-reactive group and a moiety of interest, and which reacts toform a reaction product of a modified aldehyde tagged polypeptide havingthe moiety of interest conjugated to the modified polypeptide through amodified fGly residue.

“N-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free amine group, which amine group in non-N-terminus aminoacid residues normally forms part of the covalent backbone of thepolypeptide.

“C-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free carboxyl group, which carboxyl group in non-C-terminusamino acid residues normally forms part of the covalent backbone of thepolypeptide.

By “internal site” as used in referenced to a polypeptide or an aminoacid sequence of a polypeptide means a region of the polypeptide that isnot at the N-terminus or at the C-terminus.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed, to the extent that suchcombinations embrace subject matter that are, for example, compoundsthat are stable compounds (i.e., compounds that can be made, isolated,characterized, and tested for biological activity). In addition, allsub-combinations of the various embodiments and elements thereof (e.g.,elements of the chemical groups listed in the embodiments describingsuch variables) are also specifically embraced by the present inventionand are disclosed herein just as if each and every such sub-combinationwas individually and explicitly disclosed herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides conjugates (e.g., polypeptideconjugates), hydrazinyl-substituted heteroaryl compounds for producingthe conjugates and methods of making and using the same. Embodiments ofeach are described in more detail in the sections below.

Conjugates

The present disclosure provides conjugates. By “conjugate” is meant afirst moiety that is stably associated with a second moiety. By “stablyassociated” is meant that a moiety is bound to another moiety orstructure under standard conditions. In certain embodiments, the twomoieties are bound to each other through one or more covalent bonds.

In certain embodiments, the conjugate is a polypeptide conjugate, whichincludes a polypeptide conjugated to a second moiety. As described inmore detail below, the moiety conjugated to the polypeptide can be anyof a variety of moieties such as, but not limited to, a detectablelabel, a drug, a water-soluble polymer, or a moiety for immobilizationof the polypeptide to a membrane or a surface. The moiety of interestcan be conjugated to the polypeptide at any desired site of thepolypeptide. Thus, the present disclosure provides, for example, amodified polypeptide having a moiety conjugated at a site at or near theC-terminus of the polypeptide. Other examples include a modifiedpolypeptide having a moiety conjugated at a position at or near theN-terminus of the polypeptide. Examples also include a modifiedpolypeptide having a moiety conjugated at a position between theC-terminus and the N-terminus of the polypeptide (e.g., at an internalsite of the polypeptide). Combinations of the above are also possiblewhere the modified polypeptide is conjugated to two or more moieties.

Embodiments of the present disclosure include conjugates where apolypeptide is conjugated to one or more moieties, such as 2 moieties, 3moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9moieties, or 10 or more moieties. The moieties may be conjugated to thepolypeptide at one or more sites in the polypeptide. For example, one ormore moieties may be conjugated to a single amino acid residue of thepolypeptide. In some cases, one moiety is conjugated to an amino acidresidue of the polypeptide. In other embodiments, two moieties may beconjugated to the same amino acid residue of the polypeptide. In otherembodiments, a first moiety is conjugated to a first amino acid residueof the polypeptide and a second moiety is conjugated to a second aminoacid residue of the polypeptide. Combinations of the above are alsopossible, for example where a polypeptide is conjugated to a firstmoiety at a first amino acid residue and conjugated to two othermoieties at a second amino acid residue. Other combinations are alsopossible, such as, but not limited to, a polypeptide conjugated to firstand second moieties at a first amino acid residue and conjugated tothird and fourth moieties at a second amino acid residue, etc.

The one or more amino acid residues that are conjugated to the one ormore moieties may be naturally occurring amino acids, unnatural aminoacids, or combinations thereof. For instance, the conjugate may includea moiety conjugated to a naturally occurring amino acid residue of thepolypeptide. In other instances, the conjugate may include a moietyconjugated to an unnatural amino acid residue of the polypeptide. One ormore moieties may be conjugated to the polypeptide at a single naturalor unnatural amino acid residue as described above. One or more naturalor unnatural amino acid residues in the polypeptide may be conjugated tothe moiety or moieties as described herein. For example, two (or more)amino acid residues (e.g., natural or unnatural amino acid residues) inthe polypeptide may each be conjugated to one or two moieties, such thatmultiple sites in the polypeptide are modified.

As described above, one or more moieties may be conjugated to thepolypeptide to form a moiety-polypeptide conjugate. As such, in amixture of conjugates (e.g., a reaction mixture from a conjugationreaction) the ratio of the moiety to polypeptide in the conjugate canrange from 0.1 to 100, such as 0.1 to 75, or 0.1 to 50, or 0.1 to 25, or0.1 to 10, or 0.5 to 10, or 0.5 to 9, or 0.5 to 8, or 0.5 to 7, or 0.5to 6, or 0.5 to 5, or 0.5 to 4, or 0.5 to 3, or 0.5 to 2. For instance,the ratio of the moiety to polypeptide in the conjugate can range from 1to 2. In some cases, the ratio of the moiety to polypeptide in theconjugate is 1 or 1.5. In some cases, the ratio of the moiety topolypeptide in the conjugate is measured as an average ratio from amixture of conjugates. The term “average” as used herein refers to thearithmetic mean. In certain embodiments, the moiety conjugated to thepolypeptide may be a chemical entity, such as, but not limited to, adrug or a detectable label. For example, the moiety conjugated to thepolypeptide may be a drug. In addition, in some cases, the polypeptidemay be an antibody. Thus, the conjugate can be an antibody-drugconjugate. In some embodiments, in a mixture of conjugates (e.g., areaction mixture from a conjugation reaction) the ratio of the drug toantibody in the conjugate (Drug-to-Antibody Ratio; DAR) can range from0.1 to 100, such as 0.1 to 75, or 0.1 to 50, or 0.1 to 25, or 0.1 to 10,or 0.5 to 10, or 0.5 to 9, or 0.5 to 8, or 0.5 to 7, or 0.5 to 6, or 0.5to 5, or 0.5 to 4, or 0.5 to 3, or 0.5 to 2. For instance, the DAR canrange from 1 to 2. In some cases, the DAR is 1 or 1.5. In some cases,the DAR is measured as an average ratio from a mixture of conjugates.

Although described herein in terms of a polypeptide conjugated to one ormore moieties (e.g., a chemical entity, a polypeptide, etc.),embodiments of the present disclosure also include conjugates where amoiety (e.g., a chemical entity, such as a drug or a detectable label)is conjugated to one or more other moieties (e.g., a chemical entity, apolypeptide, etc.). For example, a drug may be conjugated to one or moreother moieties (e.g., a chemical entity, a polypeptide, etc.), or inother embodiments, a detectable label may be conjugated to one or moreother moieties (e.g., a chemical entity, a polypeptide, etc.). Thus, forinstance, embodiments of the present disclosure include, but are notlimited to, the following: a conjugate of a polypeptide and a drug; aconjugate of a polypeptide and a detectable label; a conjugate of two ormore polypeptides; a conjugate of two or more drugs; a conjugate of twoof more detectable labels; a conjugate of a drug and a detectable label;a conjugate of a polypeptide, a drug and a detectable label; a conjugateof a polypeptide and two or more drugs; a conjugate of a polypeptide andtwo or more detectable labels; a conjugate of a drug and two or morepolypeptides; a conjugate of a detectable label and two or morepolypeptides; and the like.

In certain embodiments, the polypeptide and the moiety of interest areconjugated through a coupling moiety. For example, the polypeptide andthe moiety of interest may each be bound (e.g., covalently bonded) tothe coupling moiety, thus indirectly binding the polypeptide and themoiety of interest together through the coupling moiety. In some cases,the coupling moiety includes a hydrazinyl-substituted heteroarylcompound or a derivative of a hydrazinyl-substituted heteroarylcompound. In some instances, the heteroaryl group of thehydrazinyl-substituted heteroaryl compound is a 5-membered heteroarylring, where one or more atoms in the ring is other than carbon (such asN, O or S). For instance, one atom in the ring may be N, O or S. Inother instances, two atoms in the ring are N, or one atom is N andanother atom is O, or one atom is N and another atom is S, or one atomis O and another atom is S. Examples of heteroaryl groups that can beincluded in the hydrazinyl-substituted heteroaryl coupling moietyinclude, but are not limited to, imidazolyl, pyrrolyl, furanyl, thienyl(thiophenyl), and the like. For example, in certain embodiments theheteroaryl group of the hydrazinyl-substituted heteroaryl couplingmoiety is imidazolyl. In other embodiments, the the heteroaryl group ofthe hydrazinyl-substituted heteroaryl coupling moiety is pyrrolyl. Inother embodiments, the the heteroaryl group of thehydrazinyl-substituted heteroaryl coupling moiety is furanyl.

A general scheme for coupling a moiety of interest to a polypeptidethrough a hydrazinyl-substituted heteroaryl coupling moiety is shown ingeneral reaction scheme A below.

In general reaction scheme A, R may be the moiety of interest conjugatedto the polypeptide. As described herein, the moiety of interest can beany of a variety of moieties such as, but not limited to, a chemicalentity, such as a detectable label, a drug, a water-soluble polymer, ora moiety for immobilization of the polypeptide to a membrane or asurface of a substrate. R′ and R″ may each independently be any desiredsubstituent, such as, but not limited to, hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z′may be selected from CR²⁰, C, N, O and S. Z″ may be C or N. In someembodiments, one or both of Z′ and Z″ is not a carbon. R²⁰ may be anydesired substituent, such as hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.

Other coupling schemes involving a hydrazinyl-substituted heteroarylcoupling moiety are also possible. For example, another general schemefor coupling a moiety of interest to a polypeptide through ahydrazinyl-substituted heteroaryl coupling moiety is shown in thegeneral reaction scheme B below.

In general reaction scheme B, R may be the moiety of interest conjugatedto the polypeptide. As described herein, the moiety can be any of avariety of moieties such as, but not limited to, a chemical entity, suchas a detectable label, a drug, a water-soluble polymer, or a moiety forimmobilization of the polypeptide to a membrane or a surface of asubstrate. R′ and R″ may each independently be any desired substituent,such as, but not limited to, hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z′may be selected from CR²⁰, C, N, O and S. Z″ may be C or N. In someembodiments, one or both of Z′ and Z″ is not a carbon. R²⁰ may be anydesired substituent, such as hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.

Examples of coupling moieties are shown in the conjugates and compoundsdescribed in more detail below.

For example, aspects of the present disclosure include a conjugate(e.g., a polypeptide conjugate) that includes at least one modifiedamino acid residue of formula (I):

wherein:

R¹ and R² are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl;

one of R³ and R⁴ is a polypeptide and the other is selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

R⁵ and R⁶ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR⁵ and R⁶ are cyclically linked to form a 5 or 6-membered heterocyclyl;

Z¹ is selected from CR⁷, N, O and S;

Z² is C or N;

R⁷ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

L is a linker; and

W is a drug or a detectable label.

In certain embodiments, R¹ and R² are each independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In certain embodiments, R¹ is independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, R¹ is hydrogen. Incertain embodiments, R¹ is alkyl or substituted alkyl. For instance, R¹may be alkyl, such as C₁-C₆ alkyl, or C₁-C₃ alkyl (e.g., methyl). Incertain embodiments, R¹ is alkenyl or substituted alkenyl. In certainembodiments, R¹ is alkynyl or substituted alkynyl. In certainembodiments, R¹ is aryl or substituted aryl. In certain embodiments, R¹is heteroaryl or substituted heteroaryl. In certain embodiments, R¹ iscycloalkyl or substituted cycloalkyl. In certain embodiments, R¹ isheterocyclyl or substituted heterocyclyl.

In certain embodiments, R² is independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, R² is hydrogen. Incertain embodiments, R² is alkyl or substituted alkyl. For instance, R²may be alkyl, such as C₁₋₁₀ alkyl, or C₁-C₆ alkyl, or C₁-C₃ alkyl (e.g.,methyl). In certain embodiments, R² is alkenyl or substituted alkenyl.In certain embodiments, R² is alkynyl or substituted alkynyl. In certainembodiments, R² is aryl or substituted aryl. In certain embodiments, R²is heteroaryl or substituted heteroaryl. In certain embodiments, R² iscycloalkyl or substituted cycloalkyl. In certain embodiments, R² isheterocyclyl or substituted heterocyclyl.

In certain embodiments, one of R³ and R⁴ is a polypeptide and the otheris selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments, R³ is the polypeptide. In these instances, R⁴may be selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl. For example, R⁴may be hydrogen. In other embodiments, R⁴ is alkyl or substituted alkyl.For instance, R⁴ may be alkyl, such as C₁₋₁₀ alkyl, or C₁-C₆ alkyl, orC₁-C₃ alkyl (e.g., methyl). In certain embodiments, R⁴ is alkenyl orsubstituted alkenyl. In certain embodiments, R⁴ is alkynyl orsubstituted alkynyl. In certain embodiments, R⁴ is aryl or substitutedaryl. In certain embodiments, R⁴ is heteroaryl or substitutedheteroaryl. In certain embodiments, R⁴ is cycloalkyl or substitutedcycloalkyl. In certain embodiments, R⁴ is heterocyclyl or substitutedheterocyclyl.

In other embodiments, R⁴ is the polypeptide. In these instances, R³ maybe selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, cycloalkyl, substitutedcycloalkyl, heterocyclyl, and substituted heterocyclyl. For example, R³may be hydrogen. In other embodiments, R³ is alkyl or substituted alkyl.For instance, R³ may be alkyl, such as C₁₋₁₀ alkyl, or C₁-C₆ alkyl, orC₁-C₃ alkyl (e.g., methyl). In certain embodiments, R³ is alkenyl orsubstituted alkenyl. In certain embodiments, R³ is alkynyl orsubstituted alkynyl. In certain embodiments, R³ is aryl or substitutedaryl. In certain embodiments, R³ is heteroaryl or substitutedheteroaryl. In certain embodiments, R³ is cycloalkyl or substitutedcycloalkyl. In certain embodiments, R³ is heterocyclyl or substitutedheterocyclyl.

In certain embodiments, R⁵ and R⁶ are each independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, or R⁵ and R⁶ are cyclically linked to form a 5or 6-membered heterocyclyl.

In certain embodiments, R⁵ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R⁵ is hydrogen. In certain embodiments, R⁵ is alkylor substituted alkyl. In certain embodiments, R⁵ is alkenyl orsubstituted alkenyl. In certain embodiments, R⁵ is alkynyl orsubstituted alkynyl. In certain embodiments, R⁵ is alkoxy or substitutedalkoxy. In certain embodiments, R⁵ is amino or substituted amino. Incertain embodiments, R⁵ is carboxyl or carboxyl ester. In certainembodiments, R⁵ is acyl or acyloxy. In certain embodiments, R⁵ is acylamino or amino acyl. In certain embodiments, R⁵ is alkylamide orsubstituted alkylamide. In certain embodiments, R⁵ is sulfonyl. Incertain embodiments, R⁵ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R⁵ is aryl or substituted aryl. In certainembodiments, R⁵ is heteroaryl or substituted heteroaryl. In certainembodiments, R⁵ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R⁵ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R⁵ is alkyl or substituted alkyl. For example,R⁵ may be alkyl or substituted alkyl, such as, C₁₋₁₀ alkyl or C₁₋₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R⁵ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R⁵ ismethyl.

In certain embodiments, R⁶ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R⁶ is hydrogen. In certain embodiments, R⁶ is alkylor substituted alkyl. In certain embodiments, R⁶ is alkenyl orsubstituted alkenyl. In certain embodiments, R⁶ is alkynyl orsubstituted alkynyl. In certain embodiments, R⁶ is alkoxy or substitutedalkoxy. In certain embodiments, R⁶ is amino or substituted amino. Incertain embodiments, R⁶ is carboxyl or carboxyl ester. In certainembodiments, R⁶ is acyl or acyloxy. In certain embodiments, R⁶ is acylamino or amino acyl. In certain embodiments, R⁶ is alkylamide orsubstituted alkylamide. In certain embodiments, R⁶ is sulfonyl. Incertain embodiments, R⁶ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R⁶ is aryl or substituted aryl. In certainembodiments, R⁶ is heteroaryl or substituted heteroaryl. In certainembodiments, R⁶ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R⁶ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R⁶ is alkyl or substituted alkyl. For example,R⁶ may be alkyl or substituted alkyl, such as, C₁₋₁₀ alkyl or C₁₋₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R⁶ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R⁶ ismethyl.

In certain embodiments, R⁵ and R⁶ are each independently selected fromalkyl and substituted alkyl. For example, R⁵ may be alkyl or substitutedalkyl, such as, C₁₋₁₀ alkyl or C₁₋₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R⁶ may be alkyl or substitutedalkyl, such as, C₁₋₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R⁵ and R⁶ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R⁵and R⁶ are each methyl.

In certain embodiments, R⁵ and R⁶ are optionally cyclically linked toform a 5 or 6-membered heterocyclyl. In some instances, R⁵ and R⁶(together with the atoms to which they are attached) may be cyclicallylinked to form a 5-membered heterocyclyl. In some instances, R⁵ and R⁶(together with the atoms to which they are attached) may be cyclicallylinked to form a 6-membered heterocyclyl.

In certain embodiments, Z¹ is selected from CR⁷, N, O and S. In certainembodiments, Z¹ is CR⁷. In certain embodiments, Z¹ is N. In certainembodiments, Z¹ is O. In certain embodiments, Z¹ is S.

In certain embodiments, Z² is C or N. In certain embodiments, Z² is C.In certain embodiments, Z² is N.

Various combinations of Z¹ and Z² are possible. For example, in certainembodiments, Z¹ and Z² are each N. In other instances, Z¹ is CR⁷ and Z²is N. In other embodiments, Z¹ is O and Z² is C.

In certain embodiments, R⁷ (if present) is independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, R⁷ is hydrogen. Incertain embodiments, R⁷ is alkyl or substituted alkyl. For example, R⁷may be alkyl or substituted alkyl, such as, C₁₋₁₀ alkyl or C₁₋₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Incertain embodiments, R⁷ is alkenyl or substituted alkenyl. In certainembodiments, R⁷ is alkynyl or substituted alkynyl. In certainembodiments, R⁷ is alkoxy or substituted alkoxy. In certain embodiments,R⁷ is amino or substituted amino. In certain embodiments, R⁷ is carboxylor carboxyl ester. In certain embodiments, R⁷ is acyl or acyloxy. Incertain embodiments, R⁷ is acyl amino or amino acyl. In certainembodiments, R⁷ is alkylamide or substituted alkylamide. In certainembodiments, R⁷ is sulfonyl. In certain embodiments, R⁷ is thioalkoxy orsubstituted thioalkoxy. In certain embodiments, R⁷ is aryl orsubstituted aryl. In certain embodiments, R⁷ is heteroaryl orsubstituted heteroaryl. In certain embodiments, R⁷ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, R⁷ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, L is a linker. Further description of thelinker, L, is found in the disclosure herein. For example, in someembodiments, L is a linker comprising-(L¹)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e)-, where L¹, L², L³, L⁴ andL⁵ are each a linker unit, and a, b, c, d and e are each independently 0or 1, wherein the sum of a, b, c, d and e is 1 to 5, as described inmore detail below. Other linkers are also possible, as shown describedin more detail below.

In certain embodiments, W is a chemical entity, such as a drug or adetectable label. In certain embodiments, W is a drug. In certainembodiments, W is a detectable label.

In certain embodiments, the conjugate (e.g., polypeptide conjugate)includes at least one modified amino acid residue of formula (Ia):

In certain embodiments, the substituents for formula (Ia) are the sameas those described for formula (I) herein. For example, in certainembodiments, the substituents for formula (Ia), e.g., R¹, R², R³, R⁴,R⁵, R⁶, L and W, are as defined in formula (I) herein. In certainembodiments of formula (Ia), R³ is the polypeptide.

In certain embodiments, the conjugate (e.g., polypeptide conjugate)includes at least one modified amino acid residue of formula (Ib):

In certain embodiments, the substituents for formula (Ib) are the sameas those described for formula (I) herein. For example, in certainembodiments, the substituents for formula (Ib), e.g., R¹, R², R³, R⁴,R⁵, R⁶, R⁷, L and W, are as defined in formula (I) herein. In certainembodiments of formula (Ib), R³ is the polypeptide.

In certain embodiments, the conjugate (e.g., polypeptide conjugate)includes at least one modified amino acid residue of formula (Ic):

In certain embodiments, the substituents for formula (Ic) are the sameas those described for formula (I) herein. For example, in certainembodiments, the substituents for formula (Ic), e.g., R¹, R², R³, R⁴,R⁵, R⁶, L and W, are as defined in formula (I) herein. In certainembodiments of formula (Ic), R⁴ is the polypeptide.

In certain embodiments, the conjugate (e.g., polypeptide conjugate)includes at least one modified amino acid residue having the followingstructure:

where L and W are as defined in formula (I) herein, and R³ is thepolypeptide.

In certain embodiments, the conjugate (e.g., polypeptide conjugate)includes at least one modified amino acid residue having the followingstructure:

where L and W are as defined in formula (I) herein, and R³ is thepolypeptide.

In certain embodiments, the conjugate (e.g., polypeptide conjugate)includes at least one modified amino acid residue having the followingstructure:

where L and W are as defined in formula (I) herein, and R⁴ is thepolypeptide.Compounds Useful for Producing Conjugates

The present disclosure provides hydrazinyl-substituted heteroarylcompounds and derivatives thereof useful for producing the conjugates(e.g., polypeptide conjugates) described herein. In certain embodiments,the hydrazinyl-substituted heteroaryl compound (or derivative thereof)may be a coupling moiety useful for conjugation of a polypeptide to amoiety of interest (e.g., a chemical entity such as, but not limited to,a drug or detectable label). For example, the hydrazinyl-substitutedheteroaryl coupling moiety may be bound (e.g., attached through one ormore covalent bonds) to the moiety of interest (e.g., drug or detectablelabel). Reaction between the hydrazinyl-substituted heteroaryl couplingmoiety and a reactive group of the polypeptide (e.g., an fGly residue,such as a reactive aldehyde functional group of the fGly) can form oneor more covalent bonds between the hydrazinyl-substituted heteroarylcoupling moiety and the polypeptide, thus attaching the polypeptide andthe moiety of interest together through the coupling moiety.

In certain embodiments, the hydrazinyl-substituted heteroaryl compoundis a compound of formula (II):

wherein:

R is H and R⁹ is —(CR²R⁴)(NR⁶)(NHR⁵), or R⁸ is —(CR¹R³)(NR)(NHR⁶) and R⁹is H;

R¹, R², R³ and R⁴ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl;

R⁵ and R⁶ are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, orR⁵ and R⁶ are cyclically linked to form a 5 or 6-membered heterocyclyl;

Z¹ is selected from CR⁷, N, O and S;

Z² is C or N;

R⁷ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl;

L is a linker; and

W is a drug or a detectable label.

In certain embodiments, R⁸ is H and R⁹ is —(CR²R⁴)(NR⁶)(NHR⁵), or R⁸ is—(CR¹R³)(NR)(NHR⁶) and R⁹ is H. In certain embodiments, R⁸ is H and R⁹is —(CR²R⁴)(NR⁶)(NHR⁵). In certain embodiments R⁸ is —(CR¹R³)(NR)(NHR⁶)and R⁹ is H.

In certain embodiments, R¹, R², R³ and R⁴ are each independentlyselected from hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In certain embodiments, R¹ is independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, R¹ is hydrogen. Incertain embodiments, R¹ is alkyl or substituted alkyl. For instance, R¹may be alkyl, such as C₁-C₆ alkyl, or C₁-C₃ alkyl (e.g., methyl). Incertain embodiments, R¹ is alkenyl or substituted alkenyl. In certainembodiments, R¹ is alkynyl or substituted alkynyl. In certainembodiments, R¹ is aryl or substituted aryl. In certain embodiments, R¹is heteroaryl or substituted heteroaryl. In certain embodiments, R¹ iscycloalkyl or substituted cycloalkyl. In certain embodiments, R¹ isheterocyclyl or substituted heterocyclyl.

In certain embodiments, R² is independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, R² is hydrogen. Incertain embodiments, R² is alkyl or substituted alkyl. For instance, R²may be alkyl, such as C₁₋₁₀ alkyl, or C₁-C₆ alkyl, or C₁-C₃ alkyl (e.g.,methyl). In certain embodiments, R² is alkenyl or substituted alkenyl.In certain embodiments, R² is alkynyl or substituted alkynyl. In certainembodiments, R² is aryl or substituted aryl. In certain embodiments, R²is heteroaryl or substituted heteroaryl. In certain embodiments, R² iscycloalkyl or substituted cycloalkyl. In certain embodiments, R² isheterocyclyl or substituted heterocyclyl.

In certain embodiments, R³ is independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. For example, R³ may be hydrogen. In otherembodiments, R³ is alkyl or substituted alkyl. For instance, R³ may bealkyl, such as C₁₋₁₀ alkyl, or C₁-C₆ alkyl, or C₁-C₃ alkyl (e.g.,methyl). In certain embodiments, R³ is alkenyl or substituted alkenyl.In certain embodiments, R³ is alkynyl or substituted alkynyl. In certainembodiments, R³ is aryl or substituted aryl. In certain embodiments, R³is heteroaryl or substituted heteroaryl. In certain embodiments, R³ iscycloalkyl or substituted cycloalkyl. In certain embodiments, R³ isheterocyclyl or substituted heterocyclyl.

In certain embodiments, R⁴ is independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. For example, R⁴ may be hydrogen. In otherembodiments, R⁴ is alkyl or substituted alkyl. For instance, R⁴ may bealkyl, such as C₁₋₁₀ alkyl, or C₁-C₆ alkyl, or C₁-C₃ alkyl (e.g.,methyl). In certain embodiments, R⁴ is alkenyl or substituted alkenyl.In certain embodiments, R⁴ is alkynyl or substituted alkynyl. In certainembodiments, R⁴ is aryl or substituted aryl. In certain embodiments, R⁴is heteroaryl or substituted heteroaryl. In certain embodiments, R⁴ iscycloalkyl or substituted cycloalkyl. In certain embodiments, R⁴ isheterocyclyl or substituted heterocyclyl.

In certain embodiments, R⁵ and R⁶ are each independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, or R⁵ and R⁶ are cyclically linked to form a 5or 6-membered heterocyclyl.

In certain embodiments, R⁵ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R⁵ is hydrogen. In certain embodiments, R⁵ is alkylor substituted alkyl. In certain embodiments, R⁵ is alkenyl orsubstituted alkenyl. In certain embodiments, R⁵ is alkynyl orsubstituted alkynyl. In certain embodiments, R⁵ is alkoxy or substitutedalkoxy. In certain embodiments, R⁵ is amino or substituted amino. Incertain embodiments, R⁵ is carboxyl or carboxyl ester. In certainembodiments, R⁵ is acyl or acyloxy. In certain embodiments, R⁵ is acylamino or amino acyl. In certain embodiments, R⁵ is alkylamide orsubstituted alkylamide. In certain embodiments, R⁵ is sulfonyl. Incertain embodiments, R⁵ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R⁵ is aryl or substituted aryl. In certainembodiments, R⁵ is heteroaryl or substituted heteroaryl. In certainembodiments, R⁵ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R⁵ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R⁵ is alkyl or substituted alkyl. For example,R⁵ may be alkyl or substituted alkyl, such as, C₁₋₁₀ alkyl or C₁₋₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R⁵ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R⁵ ismethyl.

In certain embodiments, R⁶ is selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, R⁶ is hydrogen. In certain embodiments, R⁶ is alkylor substituted alkyl. In certain embodiments, R⁶ is alkenyl orsubstituted alkenyl. In certain embodiments, R⁶ is alkynyl orsubstituted alkynyl. In certain embodiments, R⁶ is alkoxy or substitutedalkoxy. In certain embodiments, R⁶ is amino or substituted amino. Incertain embodiments, R⁶ is carboxyl or carboxyl ester. In certainembodiments, R⁶ is acyl or acyloxy. In certain embodiments, R⁶ is acylamino or amino acyl. In certain embodiments, R⁶ is alkylamide orsubstituted alkylamide. In certain embodiments, R⁶ is sulfonyl. Incertain embodiments, R⁶ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R⁶ is aryl or substituted aryl. In certainembodiments, R⁶ is heteroaryl or substituted heteroaryl. In certainembodiments, R⁶ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R⁶ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, R⁶ is alkyl or substituted alkyl. For example,R⁶ may be alkyl or substituted alkyl, such as, C₁₋₁₀ alkyl or C₁₋₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Insome cases, R⁶ is methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R⁶ ismethyl.

In certain embodiments, R⁵ and R⁶ are each independently selected fromalkyl and substituted alkyl. For example, R⁵ may be alkyl or substitutedalkyl, such as, C₁₋₁₀ alkyl or C₁₋₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl), and R⁶ may be alkyl or substitutedalkyl, such as, C₁₋₁₀ alkyl or C₁-C₁₀ substituted alkyl (e.g., C₁-C₆alkyl or C₁-C₆ substituted alkyl). In some cases, R⁵ and R⁶ are eachindependently selected from methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, isobutyl, t-butyl, or the like. In certain cases, R⁵and R⁶ are each methyl.

In certain embodiments, R⁵ and R⁶ are optionally cyclically linked toform a 5 or 6-membered heterocyclyl. In some instances, R⁵ and R⁶(together with the atoms to which they are attached) may be cyclicallylinked to form a 5-membered heterocyclyl. In some instances, R⁵ and R⁶(together with the atoms to which they are attached) may be cyclicallylinked to form a 6-membered heterocyclyl.

In certain embodiments, Z¹ is selected from CR⁷, N, O and S. In certainembodiments, Z¹ is CR⁷. In certain embodiments, Z¹ is N. In certainembodiments, Z¹ is O. In certain embodiments, Z¹ is S.

In certain embodiments, Z² is C or N. In certain embodiments, Z² is C.In certain embodiments, Z² is N.

Various combinations of Z¹ and Z² are possible. For example, in certainembodiments, Z¹ and Z² are each N. In other instances, Z¹ is CR⁷ and Z²is N. In other embodiments, Z¹ is O and Z² is C.

In certain embodiments, R⁷ (if present) is independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, R⁷ is hydrogen. Incertain embodiments, R⁷ is alkyl or substituted alkyl. For example, R⁷may be alkyl or substituted alkyl, such as, C₁₋₁₀ alkyl or C₁₋₁₀substituted alkyl (e.g., C₁-C₆ alkyl or C₁-C₆ substituted alkyl). Incertain embodiments, R⁷ is alkenyl or substituted alkenyl. In certainembodiments, R⁷ is alkynyl or substituted alkynyl. In certainembodiments, R⁷ is alkoxy or substituted alkoxy. In certain embodiments,R⁷ is amino or substituted amino. In certain embodiments, R⁷ is carboxylor carboxyl ester. In certain embodiments, R⁷ is acyl or acyloxy. Incertain embodiments, R⁷ is acyl amino or amino acyl. In certainembodiments, R⁷ is alkylamide or substituted alkylamide. In certainembodiments, R⁷ is sulfonyl. In certain embodiments, R⁷ is thioalkoxy orsubstituted thioalkoxy. In certain embodiments, R⁷ is aryl orsubstituted aryl. In certain embodiments, R⁷ is heteroaryl orsubstituted heteroaryl. In certain embodiments, R⁷ is cycloalkyl orsubstituted cycloalkyl. In certain embodiments, R⁷ is heterocyclyl orsubstituted heterocyclyl.

In certain embodiments, L is a linker. Further description of thelinker, L, is found in the disclosure herein. For example, in someembodiments, L is a linker comprising-(L¹)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e)-, where L¹, L², L³, L⁴ andL⁵ are each a linker unit, and a, b, c, d and e are each independently 0or 1, wherein the sum of a, b, c, d and e is 1 to 5, as described inmore detail below. Other linkers are also possible, as shown describedin more detail below.

In certain embodiments, W is a chemical entity, such as a drug or adetectable label. In certain embodiments, W is a drug. In certainembodiments, W is a detectable label.

In certain embodiments, the hydrazinyl-substituted heteroaryl compoundis a compound of formula (IIa):

In certain embodiments, the substituents for formula (IIa) are the sameas those described for formula (II) herein. For example, in certainembodiments, the substituents for formula (IIa), e.g., R², R⁴, R⁵, R⁶,R⁸, L and W, are as defined in formula (II) herein.

In certain embodiments, the hydrazinyl-substituted heteroaryl compoundis a compound of formula (IIb):

In certain embodiments, the substituents for formula (IIb) are the sameas those described for formula (II) herein. For example, in certainembodiments, the substituents for formula (IIb), e.g., R², R⁴, R⁵, R⁶,R⁷, R⁸, L and W, are as defined in formula (II) herein.

In certain embodiments, the hydrazinyl-substituted heteroaryl compoundis a compound of formula (IIc):

In certain embodiments, the substituents for formula (IIc) are the sameas those described for formula (II) herein. For example, in certainembodiments, the substituents for formula (IIc), e.g., R¹, R³, R⁵, R⁶,R⁹, L and W, are as defined in formula (II) herein.

In certain embodiments, the compound is of the following structure:

where L and W are as defined in formula (II) herein.

In certain embodiments, the compound is of the following structure:

where L and W are as defined in formula (II) herein.

In certain embodiments, the compound is of the following structure:

where L and W are as defined in formula (II) herein.Linkers Useful for Conjugates and Compounds

The present disclosure provides linkers (L) useful for the conjugatesand compounds described herein, such as conjugates and compounds of eachof the formulae disclosed herein (e.g., formulae (I), (Ia), (Ib), (Ic),(II), (IIa), (IIb) and (IIc) as described herein). The linkers may beutilized to bind a coupling moiety to one or more moieties of interestand/or one or more polypeptides. In some embodiments, the linker binds acoupling moiety to either a polypeptide or a chemical entity. In someinstances, the linker binds a coupling moiety to a chemical entity(e.g., a drug or a detectable label). The linker may be bound (e.g.,covalently bound) to the coupling moiety (e.g., as described herein) atany convenient position. As described herein, the coupling moiety may beused to form a covalent attachment to a second moiety, such as apolypeptide (e.g., an antibody). Thus, the linker provided hereinattaches the chemical entity (e.g., drug or detectable label) to thepolypeptide (e.g., antibody). Stated another way, the chemical entity(e.g., drug or detectable label) is attached to the polypeptide (e.g.,antibody) through the linker.

Any convenient linker may be utilized in the subject conjugates andcompounds. In certain embodiments, L includes a group selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedamino, carboxyl, carboxyl ester, acyl amino, alkylamide, substitutedalkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl. In certain embodiments, L includes an alkyl or substitutedalkyl group. In certain embodiments, L includes an alkenyl orsubstituted alkenyl group. In certain embodiments, L includes an alkynylor substituted alkynyl group. In certain embodiments, L includes analkoxy or substituted alkoxy group. In certain embodiments, L includesan amino or substituted amino group. In certain embodiments, L includesa carboxyl or carboxyl ester group. In certain embodiments, L includesan acyl amino group. In certain embodiments, L includes an alkylamide orsubstituted alkylamide group. In certain embodiments, L includes an arylor substituted aryl group. In certain embodiments, L includes aheteroaryl or substituted heteroaryl group. In certain embodiments, Lincludes a cycloalkyl or substituted cycloalkyl group. In certainembodiments, L includes a heterocyclyl or substituted heterocyclylgroup.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol. Other linkers are also possible, as shown in theconjugates and compounds described in more detail below.

In some embodiments, L is a linker described by the formula-(L¹)_(a)-(L)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e)-, wherein L¹, L², L³, L⁴and L⁵ are each independently a linker unit, and a, b, c, d and e areeach independently 0 or 1, wherein the sum of a, b, c, d and e is 1 to5.

In certain embodiments, the sum of a, b, c, d and e is 1. In certainembodiments, the sum of a, b, c, d and e is 2. In certain embodiments,the sum of a, b, c, d and e is 3. In certain embodiments, the sum of a,b, c, d and e is 4. In certain embodiments, the sum of a, b, c, d and eis 5. In certain embodiments, a, b, c, d and e are each 1. In certainembodiments, a, b, c and d are each 1, and e is 0. In certainembodiments, a, b and c are each 1, and d and e are each 0. In certainembodiments, a and b are each 1, and c, d and e are each 0. In certainembodiments, a is 1 and b, c, d and e are each 0.

Any convenient linker units may be utilized in the subject linkers.Linker units of interest include, but are not limited to, units ofpolymers such as polyethylene glycols, polyethylenes and polyacrylates,amino acid residue(s), carbohydrate-based polymers or carbohydrateresidues and derivatives thereof, polynucleotides, alkyl groups, arylgroups, heterocycle groups, cleavable linker groups, combinationsthereof, and substituted versions thereof. In some embodiments, each ofL¹, L², L³, L⁴ and L⁵ (if present) include one or more groupsindependently selected from a polyethylene glycol, a substitutedpolyethylene glycol, an amino acid residue, an alkyl, a substitutedalkyl, an aryl, a substituted aryl, a diamine (e.g., a linking groupthat includes an alkylene diamine, such as ethylene diamine), aheterocyclic group, a substituted heterocyclic group, an acetal, adisulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, and acleavable moiety (e.g., a chemically cleavable moiety, an enzymaticallycleavable moiety (such as, but not limited to, a protease cleavablemoiety (e.g., a moiety comprising one or more amino acids), aglucuronidase cleavable moiety (e.g., a carbohydrate), a beta-lactamasecleavable moiety (e.g., a beta-lactam), etc.), a photocleavable moiety,and the like).

In some embodiments, L¹ (if present) comprises a polyethylene glycol, asubstituted polyethylene glycol, an amino acid residue, an alkyl, asubstituted alkyl, an aryl, a substituted aryl, a diamine, aheterocyclic group, a substituted heterocyclic group, an acetal, adisulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, or acleavable moiety. In some embodiments, L¹ includes a polyethyleneglycol. In some embodiments, L¹ includes a substituted polyethyleneglycol, such as, but not limited to a carboxy substituted polyethyleneglycol (e.g., a polyethylene glycol comprising a carboxy group) or anester substituted polyethylene glycol (e.g., a polyethylene glycolcomprising an ester group, such as a methyl ester). In some embodiments,L¹ includes one or more amino acid residues. In some embodiments, L¹includes an alkyl or a substituted alkyl. In some embodiments, L¹includes an aryl or a substituted aryl. In some embodiments, L¹ includesa diamine (e.g., an alkylene diamine, such as ethylene diamine). In someembodiments, L¹ includes a heterocyclic group or a substitutedheterocyclic group. In some embodiments, L¹ includes an acetal. In someembodiments, L¹ includes a disulfide. In some embodiments, L¹ includes ahydrazine. In some embodiments, L¹ includes a carbohydrate. In someembodiments, L¹ includes a beta-lactam. In some embodiments, L¹ includesan ester. In some embodiments, L¹ includes a cleavable moiety.

In some embodiments, L² (if present) comprises a polyethylene glycol, asubstituted polyethylene glycol, an amino acid residue, an alkyl, asubstituted alkyl, an aryl, a substituted aryl, a diamine, aheterocyclic group, a substituted heterocyclic group, an acetal, adisulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, or acleavable moiety. In some embodiments, L² includes a polyethyleneglycol. In some embodiments, L² includes a substituted polyethyleneglycol, such as, but not limited to a carboxy substituted polyethyleneglycol (e.g., a polyethylene glycol comprising a carboxy group) or anester substituted polyethylene glycol (e.g., a polyethylene glycolcomprising an ester group, such as a methyl ester). In some embodiments,L² includes one or more amino acid residues. In some embodiments, L²includes an alkyl or a substituted alkyl. In some embodiments, L²includes an aryl or a substituted aryl. In some embodiments, L² includesa diamine (e.g., an alkylene diamine, such as ethylene diamine). In someembodiments, L² includes a heterocyclic group or a substitutedheterocyclic group. In some embodiments, L² includes an acetal. In someembodiments, L² includes a disulfide. In some embodiments, L² includes ahydrazine. In some embodiments, L² includes a carbohydrate. In someembodiments, L² includes a beta-lactam. In some embodiments, L² includesan ester. In some embodiments, L² includes a cleavable moiety.

In some embodiments, L³ (if present) comprises a polyethylene glycol, asubstituted polyethylene glycol, an amino acid residue, an alkyl, asubstituted alkyl, an aryl, a substituted aryl, a diamine, aheterocyclic group, a substituted heterocyclic group, an acetal, adisulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, or acleavable moiety. In some embodiments, L³ includes a polyethyleneglycol. In some embodiments, L³ includes a substituted polyethyleneglycol, such as, but not limited to a carboxy substituted polyethyleneglycol (e.g., a polyethylene glycol comprising a carboxy group) or anester substituted polyethylene glycol (e.g., a polyethylene glycolcomprising an ester group, such as a methyl ester). In some embodiments,L³ includes one or more amino acid residues. In some embodiments, L³includes an alkyl or a substituted alkyl. In some embodiments, L³includes an aryl or a substituted aryl. In some embodiments, L³ includesa diamine (e.g., an alkylene diamine, such as ethylene diamine). In someembodiments, L³ includes a heterocyclic group or a substitutedheterocyclic group. In some embodiments, L³ includes an acetal. In someembodiments, L³ includes a disulfide. In some embodiments, L³ includes ahydrazine. In some embodiments, L³ includes a carbohydrate. In someembodiments, L³ includes a beta-lactam. In some embodiments, L³ includesan ester. In some embodiments, L³ includes a cleavable moiety.

In some embodiments, L⁴ (if present) comprises a polyethylene glycol, asubstituted polyethylene glycol, an amino acid residue, an alkyl, asubstituted alkyl, an aryl, a substituted aryl, a diamine, aheterocyclic group, a substituted heterocyclic group, an acetal, adisulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, or acleavable moiety. In some embodiments, L⁴ includes a polyethyleneglycol. In some embodiments, L⁴ includes a substituted polyethyleneglycol, such as, but not limited to a carboxy substituted polyethyleneglycol (e.g., a polyethylene glycol comprising a carboxy group) or anester substituted polyethylene glycol (e.g., a polyethylene glycolcomprising an ester group, such as a methyl ester). In some embodiments,L⁴ includes one or more amino acid residues. In some embodiments, L⁴includes an alkyl or a substituted alkyl. In some embodiments, L⁴includes an aryl or a substituted aryl. In some embodiments, L⁴ includesa diamine (e.g., an alkylene diamine, such as ethylene diamine). In someembodiments, L⁴ includes a heterocyclic group or a substitutedheterocyclic group. In some embodiments, L⁴ includes an acetal. In someembodiments, L¹ includes a disulfide. In some embodiments, L⁴ includes ahydrazine. In some embodiments, L⁴ includes a carbohydrate. In someembodiments, L⁴ includes a beta-lactam. In some embodiments, L⁴ includesan ester. In some embodiments, L⁴ includes a cleavable moiety.

In some embodiments, L⁵ (if present) comprises a polyethylene glycol, asubstituted polyethylene glycol, an amino acid residue, an alkyl, asubstituted alkyl, an aryl, a substituted aryl, a diamine, aheterocyclic group, a substituted heterocyclic group, an acetal, adisulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, or acleavable moiety. In some embodiments, L⁵ includes a polyethyleneglycol. In some embodiments, L⁵ includes a substituted polyethyleneglycol, such as, but not limited to a carboxy substituted polyethyleneglycol (e.g., a polyethylene glycol comprising a carboxy group) or anester substituted polyethylene glycol (e.g., a polyethylene glycolcomprising an ester group, such as a methyl ester). In some embodiments,L⁵ includes one or more amino acid residues. In some embodiments, L⁵includes an alkyl or a substituted alkyl. In some embodiments, L⁵includes an aryl or a substituted aryl. In some embodiments, L⁵ includesa diamine (e.g., an alkylene diamine, such as ethylene diamine). In someembodiments, L⁵ includes a heterocyclic group or a substitutedheterocyclic group. In some embodiments, L⁵ includes an acetal. In someembodiments, L⁵ includes a disulfide. In some embodiments, L⁵ includes ahydrazine. In some embodiments, L⁵ includes a carbohydrate. In someembodiments, L⁵ includes a beta-lactam. In some embodiments, L⁵ includesan ester. In some embodiments, L⁵ includes a cleavable moiety.

Any convenient cleavable moiety may be utilized as a cleavable linkerunit in the subject conjugates and compounds. In certain embodiments,the cleavable moiety is a para-amino-benzyloxycarbonyl group (PABC), ameta-amino-benzyloxycarbonyl group (MABC), a para-amino-benzyloxy group(PABO), a meta-amino-benzyloxy group (MABO), para-aminobenzyl (PAB), anacetal group, a disulfide, a hydrazine, a protease-cleavable moiety(e.g., e.g., a sequence of amino acids, such as a Cat B cleavablemoiety), a glucuronidase cleavable moiety (e.g., a carbohydrate), abeta-lactamase cleavable moiety (e.g., a beat-lactam), or an ester.

In some embodiments, L is a linker of the formula-(L¹)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e)-, where:-(L¹)_(a)- is -(T¹-V¹)_(a)—;-(L²)_(b)- is -(T²-V²)_(b)—;-(L³)_(c)- is -(T³-V³)_(c)—;-(L⁴)_(d)- is -(T⁴-V⁴)_(d)—; and-(L⁵)_(e)- is -(T⁵-V⁵)_(e)—;

where T¹, T², T³, T⁴ and T⁵, if present, are each tether groups;

V¹, V², V³, V⁴ and V⁵, if present, are each independently a covalentbond or a linking functional group; and

a, b, c, d and e are each independently 0 or 1, where the sum of a, b,c, d and e is 1 to 5.

Regarding the tether groups, T¹, T², T³, T⁴ and T⁵, any convenienttether groups may be utilized in the subject linkers. In someembodiments, T¹, T², T³, T⁴ and T⁵ each comprise one or more groupsindependently selected from a (C₁-C₁₂)alkyl, a substituted(C₁-C₁₂)alkyl, an (EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(h)—,4-amino-piperidinyl (4AP), para-aminobenzyl (PAB), para-amino-benzyloxy(PABO), meta-amino-benzyloxy (MABO), para-amino-benzyloxycarbonyl(PABC), meta-amino-benzyloxycarbonyl (MABC), an acetal group, adisulfide, a hydrazine, a carbohydrate, a beta-lactam, aprotease-cleavable moiety, a glucuronidase cleavable moiety, abeta-lactamase cleavable moiety, and an ester, where w is an integerfrom 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to20, and h is an integer from 1 to 12.

In some embodiments, T¹ includes one or more groups independentlyselected from a (C₁-C₁₂)alkyl, a substituted (C₁-C₁₂)alkyl, an(EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(h)—, 4AP, PAB, PABO, MABO,PABC, MABC, an acetal group, a disulfide, a hydrazine, a carbohydrate, abeta-lactam, a protease-cleavable moiety, a glucuronidase cleavablemoiety, a beta-lactamase cleavable moiety, and an ester. In someembodiments, T¹ includes a (C₁-C₁₂)alkyl or a substituted (C₁-C₁₂)alkyl,such as a (C₁-C₆)alkyl or a substituted (C₁-C₆)alkyl or a (C₁-C₃)alkylor a substituted (C₁-C₃)alkyl. In some embodiments, T¹ includes an(EDA)_(w). In some embodiments, T¹ includes a (PEG)_(n). In someembodiments, T¹ includes an (AA)_(p). In some embodiments, T¹ includes a—(CR¹³OH)_(h)—. In some embodiments, T¹ includes a 4AP. In someembodiments, T¹ includes a PAB. In some embodiments, T¹ includes a PABO.In some embodiments, T¹ includes a MABO. In some embodiments, T¹includes a PABC. In some embodiments, T¹ includes a MABC. In someembodiments, T¹ includes an acetal group. In some embodiments, T¹includes a disulfide. In some embodiments, T¹ includes a hydrazine. Insome embodiments, T¹ includes a carbohydrate. In some embodiments, T¹includes a beta-lactam. In some embodiments, T¹ includes aprotease-cleavable moiety (e.g., a sequence of one or more amino acids).In some embodiments, T¹ includes a glucuronidase cleavable moiety (e.g.,a carbohydrate). In some embodiments, T¹ includes a beta-lactamasecleavable moiety (e.g., a beta-lactam). In some embodiments, T¹ includesand an ester.

In some embodiments, T² includes one or more groups independentlyselected from a (C₁-C₁₂)alkyl, a substituted (C₁-C₁₂)alkyl, an(EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(h)—, 4AP, PAB, PABO, MABO,PABC, MABC, an acetal group, a disulfide, a hydrazine, a carbohydrate, abeta-lactam, a protease-cleavable moiety, a glucuronidase cleavablemoiety, a beta-lactamase cleavable moiety, and an ester. In someembodiments, T² includes a (C₁-C₁₂)alkyl or a substituted (C₁-C₁₂)alkyl,such as a (C₁-C₆)alkyl or a substituted (C₁-C₆)alkyl or a (C₁-C₃)alkylor a substituted (C₁-C₃)alkyl. In some embodiments, T² includes an(EDA)_(w). In some embodiments, T² includes a (PEG)_(n). In someembodiments, T² includes an (AA)_(p). In some embodiments, T² includes a—(CR¹³OH)_(h)—. In some embodiments, T² includes a 4AP. In someembodiments, T² includes a PAB. In some embodiments, T² includes a PABO.In some embodiments, T² includes a MABO. In some embodiments, T²includes a PABC. In some embodiments, T² includes a MABC. In someembodiments, T² includes an acetal group. In some embodiments, T²includes a disulfide. In some embodiments, T² includes a hydrazine. Insome embodiments, T² includes a carbohydrate. In some embodiments, T²includes a beta-lactam. In some embodiments, T² includes aprotease-cleavable moiety (e.g., a sequence of one or more amino acids).In some embodiments, T² includes a glucuronidase cleavable moiety (e.g.,a carbohydrate). In some embodiments, T² includes a beta-lactamasecleavable moiety (e.g., a beta-lactam). In some embodiments, T² includesand an ester.

In some embodiments, T³ includes one or more groups independentlyselected from a (C₁-C₁₂)alkyl, a substituted (C₁-C₁₂)alkyl, an(EDA)_(w), (PEG)_(n), (AA)_(n), —(CR¹³H)_(h)—, 4AP, PAB, PABO, MABO,PABC, MABC, an acetal group, a disulfide, a hydrazine, a carbohydrate, abeta-lactam, a protease-cleavable moiety, a glucuronidase cleavablemoiety, a beta-lactamase cleavable moiety, and an ester. In someembodiments, T³ includes a (C₁-C₁₂)alkyl or a substituted (C₁-C₁₂)alkyl,such as a (C₁-C₆)alkyl or a substituted (C₁-C₆)alkyl or a (C₁-C₃)alkylor a substituted (C₁-C₃)alkyl. In some embodiments, T³ includes an(EDA)_(w). In some embodiments, T³ includes a (PEG)_(n). In someembodiments, T³ includes an (AA)_(p). In some embodiments, T³ includes a—(CR¹³OH)_(h)—. In some embodiments, T³ includes a 4AP. In someembodiments, T³ includes a PAB. In some embodiments, T³ includes a PABO.In some embodiments, T³ includes a MABO. In some embodiments, T³includes a PABC. In some embodiments, T³ includes a MABC. In someembodiments, T³ includes an acetal group. In some embodiments, T³includes a disulfide. In some embodiments, T³ includes a hydrazine. Insome embodiments, T³ includes a carbohydrate. In some embodiments, T³includes a beta-lactam. In some embodiments, T³ includes aprotease-cleavable moiety (e.g., a sequence of one or more amino acids).In some embodiments, T³ includes a glucuronidase cleavable moiety (e.g.,a carbohydrate). In some embodiments, T³ includes a beta-lactamasecleavable moiety (e.g., a beta-lactam). In some embodiments, T³ includesand an ester.

In some embodiments, T⁴ includes one or more groups independentlyselected from a (C₁-C₁₂)alkyl, a substituted (C₁-C₁₂)alkyl, an(EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(h)—, 4AP, PAB, PABO, MABO,PABC, MABC, an acetal group, a disulfide, a hydrazine, a carbohydrate, abeta-lactam, a protease-cleavable moiety, a glucuronidase cleavablemoiety, a beta-lactamase cleavable moiety, and an ester. In someembodiments, T⁴ includes a (C₁-C₁₂)alkyl or a substituted (C₁-C₁₂)alkyl,such as a (C₁-C₆)alkyl or a substituted (C₁-C₆)alkyl or a (C₁-C₃)alkylor a substituted (C₁-C₃)alkyl. In some embodiments, T⁴ includes an(EDA)_(w). In some embodiments, T⁴ includes a (PEG)_(n). In someembodiments, T⁴ includes an (AA)_(p). In some embodiments, T⁴ includes a—(CR¹³H)_(h)—. In some embodiments, T⁴ includes a 4AP. In someembodiments, T⁴ includes a PAB. In some embodiments, T⁴ includes a PABO.In some embodiments, T⁴ includes a MABO. In some embodiments, T⁴includes a PABC. In some embodiments, T⁴ includes a MABC. In someembodiments, T⁴ includes an acetal group. In some embodiments, T⁴includes a disulfide. In some embodiments, T⁴ includes a hydrazine. Insome embodiments, T⁴ includes a carbohydrate. In some embodiments, T⁴includes a beta-lactam. In some embodiments, T⁴ includes aprotease-cleavable moiety (e.g., a sequence of one or more amino acids).In some embodiments, T⁴ includes a glucuronidase cleavable moiety (e.g.,a carbohydrate). In some embodiments, T⁴ includes a beta-lactamasecleavable moiety (e.g., a beta-lactam). In some embodiments, T⁴ includesand an ester.

In some embodiments, T⁵ includes one or more groups independentlyselected from a (C₁-C₁₂)alkyl, a substituted (C₁-C₁₂)alkyl, an(EDA)_(w), (PEG)_(n), (AA)_(p), —(CR¹³OH)_(h)—, 4AP, PAB, PABO, MABO,PABC, MABC, an acetal group, a disulfide, a hydrazine, a carbohydrate, abeta-lactam, a protease-cleavable moiety, a glucuronidase cleavablemoiety, a beta-lactamase cleavable moiety, and an ester. In someembodiments, T⁵ includes a (C₁-C₁₂)alkyl or a substituted (C₁-C₁₂)alkyl,such as a (C₁-C₆)alkyl or a substituted (C₁-C₆)alkyl or a (C₁-C₃)alkylor a substituted (C₁-C₃)alkyl. In some embodiments, T⁵ includes an(EDA)_(w). In some embodiments, T⁵ includes a (PEG)_(n). In someembodiments, T⁵ includes an (AA)_(p). In some embodiments, T⁵ includes a—(CR¹³H)_(h)—. In some embodiments, T⁵ includes a 4AP. In someembodiments, T⁵ includes a PAB. In some embodiments, T⁵ includes a PABO.In some embodiments, T⁵ includes a MABO. In some embodiments, T⁵includes a PABC. In some embodiments, T⁵ includes a MABC. In someembodiments, T⁵ includes an acetal group. In some embodiments, T⁵includes a disulfide. In some embodiments, T⁵ includes a hydrazine. Insome embodiments, T⁵ includes a carbohydrate. In some embodiments, T⁵includes a beta-lactam. In some embodiments, T⁵ includes aprotease-cleavable moiety (e.g., a sequence of one or more amino acids).In some embodiments, T⁵ includes a glucuronidase cleavable moiety (e.g.,a carbohydrate). In some embodiments, T⁵ includes a beta-lactamasecleavable moiety (e.g., a beta-lactam). In some embodiments, T⁵ includesand an ester.

In some instances, T¹, T², T³, T⁴ and T⁵ may each include a combinationof one or more tether groups described above, such as, but not limitedto combinations such as (AA)_(p)-MABO, (AA)_(p)-MABC, (AA)_(p)-PABO,(AA)_(p)-PABC, MABO-(AA)_(p), MABC-(AA)_(p), PABO-(AA)_(p),PABC-(AA)_(p), (AA)_(p)-MABO-(AA)_(p), (AA)_(p)-MABC-(AA)_(p),(AA)_(p)-PABO-(AA)_(p), and (AA)_(p)-PABC-(AA)_(p).

For example, T¹ may include a tether group, such as (AA)_(p)-MABO. Insome embodiments, T¹ includes (AA)_(p)-MABC. In some embodiments, T¹includes (AA)_(p)-PABO. In some embodiments, T¹ includes (AA)_(p)-PABC.In some embodiments, T¹ includes MABO-(AA)_(p). In some embodiments, T¹includes MABC-(AA)_(p). In some embodiments, T¹ includes PABO-(AA)_(p).In some embodiments, T¹ includes PABC-(AA)_(p). In some embodiments, T¹includes (AA)_(p)-MABO-(AA)_(p). In some embodiments, T¹ includes(AA)_(p)-MABC-(AA)_(p). In some embodiments, T¹ includes(AA)_(p)-PABO-(AA)_(p). In some embodiments, T¹ includes(AA)_(p)-PABC-(AA)_(p).

In some embodiments, T² includes a tether group, such as (AA)_(p)-MABO.In some embodiments, T² includes (AA)_(p)-MABC. In some embodiments, T²includes (AA)_(p)-PABO. In some embodiments, T² includes (AA)_(p)-PABC.In some embodiments, T² includes MABO-(AA)_(p). In some embodiments, T²includes MABC-(AA)_(p). In some embodiments, T² includes PABO-(AA)_(p).In some embodiments, T² includes PABC-(AA)_(p). In some embodiments, T²includes (AA)_(p)-MABO-(AA)_(p). In some embodiments, T² includes(AA)_(p)-MABC-(AA)_(p). In some embodiments, T² includes(AA)_(p)-PABO-(AA)_(p). In some embodiments, T² includes(AA)_(p)-PABC-(AA)_(p).

In some embodiments, T³ includes a tether group, such as (AA)_(p)-MABO.In some embodiments, T³ includes (AA)_(p)-MABC. In some embodiments, T³includes (AA)_(p)-PABO. In some embodiments, T³ includes (AA)_(p)-PABC.In some embodiments, T³ includes MABO-(AA)_(p). In some embodiments, T³includes MABC-(AA)_(p). In some embodiments, T³ includes PABO-(AA)_(p).In some embodiments, T³ includes PABC-(AA)_(p). In some embodiments, T³includes (AA)_(p)-MABO-(AA)_(p). In some embodiments, T³ includes(AA)_(p)-MABC-(AA)_(p). In some embodiments, T³ includes(AA)_(p)-PABO-(AA)_(p). In some embodiments, T³ includes(AA)_(p)-PABC-(AA)_(p).

In some embodiments, T⁴ includes a tether group, such as (AA)_(p)-MABO.In some embodiments, T⁴ includes (AA)_(p)-MABC. In some embodiments, T⁴includes (AA)_(p)-PABO. In some embodiments, T⁴ includes (AA)_(p)-PABC.In some embodiments, T⁴ includes MABO-(AA)_(p). In some embodiments, T⁴includes MABC-(AA)_(p). In some embodiments, T⁴ includes PABO-(AA)_(p).In some embodiments, T⁴ includes PABC-(AA)_(p). In some embodiments, T⁴includes (AA)_(p)-MABO-(AA)_(p). In some embodiments, T⁴ includes(AA)_(p)-MABC-(AA)_(p). In some embodiments, T⁴ includes(AA)_(p)-PABO-(AA)_(p). In some embodiments, T⁴ includes(AA)_(p)-PABC-(AA)_(p).

In some embodiments, T⁵ includes a tether group, such as (AA)_(p)-MABO.In some embodiments, T⁵ includes (AA)_(p)-MABC. In some embodiments, T⁵includes (AA)_(p)-PABO. In some embodiments, T⁵ includes (AA)_(p)-PABC.In some embodiments, T⁵ includes MABO-(AA)_(p). In some embodiments, T⁵includes MABC-(AA)_(p). In some embodiments, T⁵ includes PABO-(AA)_(p).In some embodiments, T⁵ includes PABC-(AA)_(p). In some embodiments, T⁵includes (AA)_(p)-MABO-(AA)_(p). In some embodiments, T⁵ includes(AA)_(p)-MABC-(AA)_(p). In some embodiments, T⁵ includes(AA)_(p)-PABO-(AA)_(p). In some embodiments, T⁵ includes(AA)_(p)-PABC-(AA)_(p).

As described above, the tether group may include an ethylene diamine(EDA) moiety, e.g., an EDA containing tether group. In certainembodiments, (EDA)_(w) includes one or more EDA moieties, such as wherew is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5 or 6. Theethylene diamine (EDA) moieties may optionally be substituted at one ormore convenient positions with any convenient substituents, e.g., withan alkyl, a substituted alkyl, an acyl, a substituted acyl, an aryl or asubstituted aryl. In certain embodiments, the EDA moiety is described bythe structure:

where q is an integer from 1 to 6 and r is 0 or 1 and each R¹² isindependently selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or any two adjacent R¹²groups may be cyclically linked to form a piperazinyl ring.

In certain embodiments, q is 1, 2, 3, 4, 5 or 6. In certain embodiments,q is 1 and r is 0. In certain embodiments, q is 1 and r is 1. In certainembodiments, q is 2 and r is 0. In certain embodiments, q is 2 and r is1.

In certain embodiments, each R¹² is independently selected fromhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or wherein two adjacent R¹²groups are cyclically linked to form a piperazinyl ring. In certainembodiments, each R¹² is independently selected from hydrogen, an alkyl,a substituted alkyl, an aryl and a substituted aryl. In certainembodiments, any two adjacent R¹² groups of the EDA may be cyclicallylinked, e.g., to form a piperazinyl ring. In certain embodiments, q is 1and the two adjacent R¹² groups are a cyclically linked alkenylenegroup, thus forming a piperazinyl ring. In certain embodiments, q is 1and the adjacent R¹² groups are selected from hydrogen, an alkyl (e.g.,methyl) and a substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OHor propyl-OH).

As described above, the tether group may include a 4-amino-piperidinyl(4AP) moiety (also referred to as a piperidin-4-amino (P4A) moiety). The4AP moiety may optionally be substituted at one or more convenientpositions with any convenient substituents, e.g., with an alkyl, asubstituted alkyl, a polyethylene glycol, a substituted polyethyleneglycol, an acyl, a substituted acyl, an aryl or a substituted aryl. Incertain embodiments, the 4AP moiety is described by the structure:

where R¹⁴ is selected from hydrogen, alkyl, substituted alkyl, apolyethylene glycol moiety (e.g., a polyethylene glycol or a substitutedpolyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments, each R¹⁴ is independently selected fromhydrogen, alkyl, substituted alkyl, polyethylene glycol, substitutedpolyethylene glycol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl.

In certain embodiments, R¹⁴ is a polyethylene glycol. In certainembodiments, R¹⁴ is a substituted polyethylene glycol, such as, but notlimited to a carboxy substituted polyethylene glycol (e.g., apolyethylene glycol that includes a carboxy substituent, such as acarboxy substituent at one end of the polyethylene glycol) or an estersubstituted polyethylene glycol (e.g., a polyethylene glycol thatincludes an ester substituent, such as a methyl ester, such as an estersubstituent as one end of the polyethylene glycol). The polyethyleneglycol or substituted polyethylene glycol substituent at the R¹⁴position can include one or more polyethylene glycol subunits, such as 1to 50, or 1 to 40, or 1 to 30, or 1 to 20, or 1 to 12, or 1 to 10, or 1to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 polyethylene glycol subunits. In some instances, thepolyethylene glycol or substituted polyethylene glycol substituent atthe R¹⁴ position includes 3 polyethylene glycol subunits. In someinstances, the polyethylene glycol or substituted polyethylene glycolsubstituent at the R¹⁴ position includes 6 polyethylene glycol subunits.In some instances, the polyethylene glycol or substituted polyethyleneglycol substituent at the R¹⁴ position includes 8 polyethylene glycolsubunits. In some instances, the polyethylene glycol or substitutedpolyethylene glycol substituent at the R¹⁴ position includes 10polyethylene glycol subunits. In some instances, the polyethylene glycolor substituted polyethylene glycol substituent at the R¹⁴ positionincludes 12 polyethylene glycol subunits.

As described above, the tether group may include a (PEG)_(n), group,where PEG is a polyethylene glycol or a substituted polyethylene glycol.In certain embodiments, (PEG)_(n), is described by the structure:

where n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30,from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, nis 3. In some instances, n is 6. In some instances, n is 8. In someinstances, n is 10. In some instances, n is 12.

In certain embodiments, a tether group includes (AA)_(p), where AA is anamino acid residue. Any convenient amino acids may be utilized. Aminoacids of interest include but are not limited to, L- and D-amino acids,naturally occurring amino acids such as any of the 20 primaryalpha-amino acids and beta-alanine, non-naturally occurring amino acids(e.g., amino acid analogs), such as a non-naturally occurringalpha-amino acid or a non-naturally occurring beta-amino acid, etc. Incertain embodiments, p is 1. In certain embodiments, p is an integerfrom 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20.

In certain embodiments, a tether group includes a moiety described bythe formula —(CR¹³OH)_(h)—, where h is 0 or n is an integer from 1 to50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 orfrom 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

In certain embodiments, R¹³ is selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Incertain embodiments, each R¹³ is independently selected from hydrogen,alkyl, substituted alkyl, aryl, and substituted aryl.

In certain embodiments, R¹³ is hydrogen. In certain embodiments, R¹³ isalkyl or substituted alkyl. In certain embodiments, R¹³ is alkenyl orsubstituted alkenyl. In certain embodiments, R¹³ is alkynyl orsubstituted alkynyl. In certain embodiments, R¹³ is alkoxy orsubstituted alkoxy. In certain embodiments, R¹³ is amino or substitutedamino. In certain embodiments, R¹³ is carboxyl or carboxyl ester. Incertain embodiments, R¹³ is acyl or acyloxy. In certain embodiments, R¹³is acyl amino or amino acyl. In certain embodiments, R¹³ is alkylamideor substituted alkylamide. In certain embodiments, R¹³ is sulfonyl. Incertain embodiments, R¹³ is thioalkoxy or substituted thioalkoxy. Incertain embodiments, R¹³ is aryl or substituted aryl. In certainembodiments, R¹³ is heteroaryl or substituted heteroaryl. In certainembodiments, R¹³ is cycloalkyl or substituted cycloalkyl. In certainembodiments, R¹³ is heterocyclyl or substituted heterocyclyl. In certainembodiments, R¹³ is selected from hydrogen, an alkyl, a substitutedalkyl, an aryl, and a substituted aryl.

In some embodiments, a tether group includes a para-aminobenzyl (PAB)group described by the following structure:

In some embodiments, a tether group includes a para-aminobenzoyloxy(PABO) group described by the following structure:

In some embodiments, a tether group includes apara-amino-benzyloxycarbonyl (PABC) group described by the followingstructure:

In some embodiments of the PAB, PABO, and PABC tether groups shownabove, R¹⁵ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments ofPAB, PABO, and PABC, R¹⁵ is selected from hydrogen, an alkyl, asubstituted alkyl, an aryl and a substituted aryl. In certainembodiments of PAB, PABO, and PABC, R¹⁵ is selected from hydrogen, analkyl (e.g., methyl) and a substituted alkyl (e.g., lower alkyl-OH, suchas ethyl-OH or propyl-OH). In some embodiments, any of the PAB, PABO,and PABC tether groups shown above may be further substituted with oneor more convenient aryl and/or alkyl substituents. In certainembodiments of PAB, PABO, and PABC, R¹⁵ is hydrogen. The divalent PAB,PABO, and PABC tether groups may be covalently bound to adjacentmoieties via any convenient chemistries.

In some embodiments, a tether group includes a meta-amino-benzyloxy(MABO) group described by the following structure:

In some embodiments, a tether group includes ameta-amino-benzyloxycarbonyl (MABC) group described by the followingstructure:

In some embodiments of the MABO and MABC tether groups shown above, R¹⁵is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In certain embodiments ofMABO and MABC, R¹⁵ is selected from hydrogen, an alkyl, a substitutedalkyl, an aryl and a substituted aryl. In certain embodiments of MABOand MABC, R¹⁵ is selected from hydrogen, an alkyl (e.g., methyl) and asubstituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).In some embodiments, any of the MABO and MABC tether groups shown abovemay be further substituted with one or more convenient aryl and/or alkylsubstituents. In certain embodiments of MABO and MABC, R¹⁵ is hydrogen.The divalent MABO and MABC tether groups may be covalently bound toadjacent moieties via any convenient chemistries.

In some embodiments of the MABO and MABC tether groups shown above, R¹⁶is selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In some embodiments of theMABO and MABC tether groups shown above, R¹⁶ is a carbohydrate orcarbohydrate derivative, such as, but not limited to, α-D-glucose orβ-D-glucose.

Regarding V¹, V², V³, V⁴ and V⁵, any convenient linking functionalgroups may be utilized in the subject linkers. Linking functional groupsof interest include, but are not limited to, amino, carbonyl, amido,oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl,thio, oxy, phospho, phosphoramidate, thiophosphoramidate, and the like.In some embodiments, V¹, V², V³, V⁴ and V⁵ are each independentlyselected from the group consisting of a covalent bond, —CO—, —NR¹¹—,—CONR¹¹—, —NR¹¹CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—,—SO₂NR¹¹—, —NR¹¹SO₂— and —P(O)OH—.

In some embodiments, V¹ is selected from a covalent bond, —CO—, —NR¹¹—,—CONR¹¹—, —NR¹¹CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—,—SO₂NR¹¹—, —NR¹SO₂— and —P(O)OH—. In some embodiments, V¹ is a covalentbond. In some embodiments, V¹ is —CO—. In some embodiments, V¹ is—NR¹¹—. In some embodiments, V¹ is —CONR¹¹—. In some embodiments, V¹ is—NR¹¹CO—. In some embodiments, V¹ is —C(O)O—. In some embodiments, V¹ is—OC(O)—. In some embodiments, V¹ is —O—. In some embodiments, V¹ is —S—.In some embodiments, V¹ is —S(O)—. In some embodiments, V¹ is —SO₂—. Insome embodiments, V¹ is —SO₂NR¹¹—. In some embodiments, V¹ is —NR¹¹SO₂—.In some embodiments, V¹ is —P(O)OH—.

In some embodiments, V² is selected from a covalent bond, —CO—, —NR¹¹—,—CONR¹¹—, —NR¹CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—,—NR¹SO₂— and —P(O)OH—. In some embodiments, V² is a covalent bond. Insome embodiments, V² is —CO—. In some embodiments, V² is —NR¹¹. In someembodiments, V² is —CONR¹¹. In some embodiments, V² is —NR¹CO¹¹. In someembodiments, V² is —C(O)O—. In some embodiments, V² is —OC(O)—. In someembodiments, V² is —O—. In some embodiments, V² is —S—. In someembodiments, V² is —S(O)—. In some embodiments, V² is —SO₂—. In someembodiments, V² is —SO₂NR¹¹—. In some embodiments, V² is —NR¹¹SO₂—. Insome embodiments, V² is —P(O)OH—.

In some embodiments, V³ is selected from a covalent bond, —CO—, —NR¹¹—,—CONR¹¹—, —NR¹CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—,—NR¹SO₂— and —P(O)OH—. In some embodiments, V³ is a covalent bond. Insome embodiments, V³ is —CO—. In some embodiments, V³ is —NR¹¹—. In someembodiments, V³ is —CONR¹¹—. In some embodiments, V³ is —NR¹¹CO—. Insome embodiments, V³ is —C(O)O—. In some embodiments, V³ is —OC(O)—. Insome embodiments, V³ is —O—. In some embodiments, V³ is —S—. In someembodiments, V³ is —S(O)—. In some embodiments, V³ is —SO₂—. In someembodiments, V³ is —SO₂NR¹¹—. In some embodiments, V³ is —NR¹¹SO₂—. Insome embodiments, V³ is —P(O)OH—.

In some embodiments, V⁴ is selected from a covalent bond, —CO—, —NR¹¹—,—CONR¹¹—, —NR¹CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—, —SO₂NR¹¹—,—NR¹SO₂— and —P(O)OH—. In some embodiments, V⁴ is a covalent bond. Insome embodiments, V⁴ is —CO—. In some embodiments, V⁴ is —NR¹¹—. In someembodiments, V⁴ is —CONR¹¹—. In some embodiments, V⁴ is —NR¹¹CO—. Insome embodiments, V⁴ is —C(O)O—. In some embodiments, V⁴ is —OC(O)—. Insome embodiments, V⁴ is —O—. In some embodiments, V⁴ is —S—. In someembodiments, V⁴ is —S(O)—. In some embodiments, V⁴ is —SO₂—. In someembodiments, V⁴ is —SO₂NR¹¹—. In some embodiments, V⁴ is —NR¹¹SO₂—. Insome embodiments, V⁴ is —P(O)OH—.

In some embodiments, V⁵ is selected from a covalent bond, —CO—, —NR¹¹—,—CONR¹¹—, —NR¹¹CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—,—SO₂NR¹¹—, —NR¹¹SO₂— and —P(O)OH—. In some embodiments, V⁵ is a covalentbond. In some embodiments, V⁵ is —CO—. In some embodiments, V⁵ is—NR¹¹—. In some embodiments, V⁵ is —CONR¹¹—. In some embodiments, V⁵ is—NR¹¹CO—. In some embodiments, V⁵ is —C(O)O—. In some embodiments, V⁵ is—OC(O)—. In some embodiments, V⁵ is —O—. In some embodiments, V⁵ is —S—.In some embodiments, V⁵ is —S(O)—. In some embodiments, V⁵ is —SO₂—. Insome embodiments, V⁵ is —SO₂NR¹¹—. In some embodiments, V⁵ is —NR¹¹SO₂—.In some embodiments, V⁵ is —P(O)OH—.

In some embodiments of V¹, V², V³, V⁴ and V⁵, each R¹ is independentlyselected from hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl. In some embodiments, eachR¹¹ is independently selected from hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments, R¹¹ is hydrogen. In certain embodiments, R¹¹ isalkyl or substituted alkyl. In certain embodiments, R¹¹ is alkenyl orsubstituted alkenyl. In certain embodiments, R¹¹ is alkynyl orsubstituted alkynyl. In certain embodiments, R¹¹ is alkoxy orsubstituted alkoxy. In certain embodiments, R¹¹ is carboxyl or carboxylester. In certain embodiments, R¹¹ is acyl or acyloxy. In certainembodiments, R¹¹ is acyl amino or amino acyl. In certain embodiments,R¹¹ is alkylamide or substituted alkylamide. In certain embodiments, R¹¹is sulfonyl. In certain embodiments, R¹¹ is thioalkoxy or substitutedthioalkoxy. In certain embodiments, R¹¹ is aryl or substituted aryl. Incertain embodiments, R¹¹ is heteroaryl or substituted heteroaryl. Incertain embodiments, R¹¹ is cycloalkyl or substituted cycloalkyl. Incertain embodiments, R¹¹ is heterocyclyl or substituted heterocyclyl.

In certain embodiments, T¹, T², T³, T⁴ and T⁵ and V¹, V², V³, V⁴ and V⁵are selected from the following table, e.g., one row of the followingtable:

T¹ V¹ T² V² T³ V³ T⁴ V⁴ T⁵ V⁵ (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h)—CONR¹¹— (C₁-C₁₂)alkyl —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—C₁-C₁₂)alkyl —CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— — — — —— — (C₁-C₁₂)alkyl —CO— (AA)_(p) — — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—(PEG)_(n) —NR¹¹— — — — — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(PEG)_(n) —NR¹¹— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— — — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (PEG)_(n) —CO— (EDA)_(w) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w)— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — — — — —(C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —SO₂— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (CR¹³OH)_(h) —CO— — — — — — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹— (PEG)_(n) —CO— — — (C₁-C₁₂)alkyl—CONR¹¹— substituted —NR¹¹— (PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl(C₁-C₁₂)alkyl —SO2— (C₁-C₁₂)alkyl —CO— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (C₁-C₁₂)alkyl — (CR¹³OH)_(h) —CONR¹¹— — — — — (C₁-C₁₂)alkyl—CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) —NR¹¹— — — (C₁-C₁₂)alkyl—CO— (AA)_(p) —NR¹¹— (PEG)_(n) —P(O)OH— (AA)_(p) — — — (C₁-C₁₂)alkyl—CO— (EDA)_(w) —CO— (AA)_(p) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO—(CR¹³OH)_(h) —CONR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — (C₁-C₁₂)alkyl—CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — —CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹—(C₁-C₁₂)alkyl —NR¹¹— — —CO— (C₁-C₁₂)alkyl —NR¹¹— — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —(C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— — — — — (C₁-C₁₂)alkyl—CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —CO— MABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— MABC — (AA)_(p) — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— (AA)_(p) - —NR¹¹— (C₁-C₁₂) alkyl —CO— PABC(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) - — — —PABC-(AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)— PABC-(AA)_(p) — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO— — — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) — PABO —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — (AA)_(p) —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABO — — — — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— PABC — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO—MABC — (AA)_(p) — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —PABC —NR¹¹— — — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p)— PABC — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) —PABC-(AA)_(p) — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p)— — —

As described above, in some embodiments, L is a linker comprising-(L¹)_(a)-(L²)_(b)-(L³)_(c)-(L⁴)_(d)-(L⁵)_(e), where -(L¹)_(a)- is-(T¹-V¹)_(a); -(L²)_(b)- is -(T²-V²)_(b)—; -(L³)_(c)- is -(T³-V³)_(c)—;-(L⁴)_(d)- is -(T⁴-V⁴)_(d)—; and -(L⁵)_(e)- is -(T⁵-V⁵)_(e)—.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is absent, V⁴ is absent,T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(EDA)_(w), V² is —CO—, T³ is (CR¹³OH)_(h), V³ is —CONR¹—, T⁴ is(C₁-C₁₂)alkyl, V⁴ is —CO—, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is absent, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —CO—, T³ is absent, V³ is absent, T⁴ is absent, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is absent, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —NR¹¹—, T³ is absent, V³ is absent, T⁴ is absent, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —NR¹¹—, T⁴ is absent, V⁴ is absent,T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(EDA)_(w), V² is —CO—, T³ is absent, V³ is absent, T⁴ is absent, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(C₁-C₁₂)alkyl, V² is —NR¹¹—, T³ is absent, V³ is absent, T⁴ is absent,V⁴ is absent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —CO—, T³ is (EDA)_(w), V³ is absent, T⁴ is absent, V⁴is absent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(EDA)_(w), V² is absent, T³ is absent, V³ is absent, T⁴ is absent, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ is absent, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(EDA)_(w), V² is —CO—, T³ is (CR¹³OH)_(h), V³ is —CO—, T⁴ is (AA)_(p),V⁴ is absent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)_(p), V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —SO₂-T⁴ is (AA)_(p), V⁴ is absent,T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(CR¹³⁰H)_(h), V² is —CO—, T³ is absent, V³ is absent, T⁴ is absent, V⁴is absent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(EDA)_(w), V² is —CO—, T³ is (CR¹³OH)_(h), V³ is —CONR¹¹—, T⁴ is(PEG)_(n), V⁴ is —CO—, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² issubstituted (C₁-C₁₂)alkyl, V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴is absent, V⁴ is absent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —SO₂—, T² is(C₁-C₁₂)alkyl, V² is —CO—, T³ is absent, V³ is absent, T⁴ is absent, V⁴is absent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(C₁-C₁₂)alkyl, V² is absent, T³ is (CR¹³OH)_(h), V³ is —CONR¹¹—, T⁴ isabsent, V⁴ is absent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is (AA)_(p), V⁴ is —NR¹¹—,T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —P(O)OH—, T⁴ is (AA)_(p), V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(EDA)_(w), V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ is absent, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(EDA)_(w), V² is —CO—, T³ is (CR¹³OH)_(h), V³ is —CONR¹¹—, T⁴ is(C₁-C₁₂)alkyl, V⁴ is —CO—, T⁵ is (AA)_(p), and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(C₁-C₁₂)alkyl, V² is —NR¹¹—, T³ is absent, V³ is —CO—, T⁴ is absent, V⁴is absent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(C₁-C₁₂)alkyl, V² is —NR¹¹—, T³ is absent, V³ is —CO—, T⁴ is(C₁-C₁₂)alkyl, V⁴ is —NR¹¹—, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is(EDA)_(w), V² is —CO—, T³ is (CR¹³OH)_(h), V³ is —CONR¹¹—, T⁴ is(PEG)_(n), V⁴ is —CO—, T⁵ is (AA)_(p), and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is 4AP, V²is —CO—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is absent, V⁴ is absent, T⁵is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is 4AP, V²is —CO—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is MABO, V⁴ is absent, T⁵is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is MABC, V⁴ is absent, T⁵is (AA)_(p), and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is (AA)_(p)-PABC, V⁴ is—NR¹¹—, T⁵ is (C₁-C₁₂)alkyl, and V⁵ is —CO—.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is PABC, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is PABO, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is (AA)_(p)-PABC-(AA)_(p),V⁴ is absent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG)_(n), V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is PABC-(AA)_(p), and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ is PABO, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is 4AP, V²is —CO—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is PABO, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ is MABO, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ is PABC, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ is MABC, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ is PABC, V⁴ isabsent, T⁵ is (AA)_(p), and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is(PEG)_(n), V² is —CO—, T³ is MABO, V³ is absent, T⁴ is absent, V⁴ isabsent, T⁵ is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V is —NR¹¹—, T³ is (PEG), V³ is —CO—, T⁴ is PABO, V⁴ is absent, T⁵ isabsent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is (AA)_(p),V² is —NR¹¹—, T³ is (PEG), V³ is —CO—, T⁴ is PABC, V⁴ is absent, T⁵ isabsent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is (PEG),V² is —CO—, T³ is MABC, V³ is absent, T⁴ is (AA)_(p), V⁴ is absent, T⁵is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CONR¹—, T² is (PEG),V² is —CO—, T³ is (AA)_(p), V³ is absent, T⁴ is PABC, V⁴ is —NR¹¹—, T⁵is absent, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is 4AP, V²is —CO—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is PABC, and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is 4AP, V²is —CO—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is PABC-(AA)_(p), and V⁵ is absent.

In certain embodiments, T¹ is (C₁-C₁₂)alkyl, V¹ is —CO—, T² is 4AP, V²is —CO—, T³ is (C₁-C₁₂)alkyl, V³ is —CO—, T⁴ is (AA)_(p), V⁴ is absent,T⁵ is absent, and V⁵ is absent.

In some embodiments, the linker is described by one of the followingstructures:

wherein:

each f is independently 0 or an integer from 1 to 12;

each n is independently 0 or an integer from 1 to 30;

each y is independently 0 or an integer from 1 to 20;

each h is independently 0 or an integer from 1 to 12;

each p is independently 0 or an integer from 1 to 20;

each R is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

each R′ is independently selected from hydrogen, a sidechain group of anamino acid, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl.

In certain embodiments of the linker structures depicted above, each fis independently 0 or an integer from 1 to 12; each n is independently 0or an integer from 1 to 30; each y is independently 0 or an integer from1 to 20; each h is independently 0 or an integer from 1 to 12; each p isindependently 0 or an integer from 1 to 20.

In certain embodiments of the linker structures depicted above, each Ris independently hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In certain embodiments of the linker structures depicted above, each R′is independently hydrogen, a sidechain of an amino acid, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments of the linker structures depicted above, each fis independently 0, 1, 2, 3, 4, 5 or 6; each n is independently 0, 1, 2,3, 4, 5 or 6; each y is independently 0, 1, 2, 3, 4, 5 or 6; each h isindependently 0, 1, 2, 3, 4, 5 or 6; and each p is independently 0, 1,2, 3, 4, 5 or 6. In certain embodiments of the linker structuresdepicted above, each R is independently H, methyl or —(CH₂)_(m)—OH wherem is 1, 2, 3, 4, 5 or 6 (e.g., 2).

In certain embodiments, the linker includes a cleavable group, e.g., asdescribed in the following structures:

wherein:

each f is independently 0 or an integer from 1 to 12;

each n is independently 0 or an integer from 1 to 30;

each y is independently 0 or an integer from 1 to 20;

each h is independently 0 or an integer from 1 to 12;

each p is independently 0 or an integer from 1 to 20;

each R is independently selected from hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

each R′ is independently selected from hydrogen, a sidechain group of anamino acid, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino,amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy,substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl.

In certain embodiments of the linker structures depicted above, each fis independently 0 or an integer from 1 to 12; each n is independently 0or an integer from 1 to 30; each y is independently 0 or an integer from1 to 20; each h is independently 0 or an integer from 1 to 12; each p isindependently 0 or an integer from 1 to 20.

In certain embodiments of the linker structures depicted above, each Ris independently hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.

In certain embodiments of the linker structures depicted above, each R′is independently hydrogen, a sidechain of an amino acid, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In certain embodiments of the linker structures depicted above, each fis independently 0, 1, 2, 3, 4, 5 or 6; each n is independently 0, 1, 2,3, 4, 5 or 6; each y is independently 0, 1, 2, 3, 4, 5 or 6; each h isindependently 0, 1, 2, 3, 4, 5 or 6; and each p is independently 0, 1,2, 3, 4, 5 or 6. In certain embodiments of the linker structuresdepicted above, each R is independently H, methyl or —(CH₂)_(m)—OH wherem is 1, 2, 3, 4, 5 or 6 (e.g., 2).

Any of the chemical entities, linkers and coupling moieties set forth inthe structures above may be adapted for use in the subject compounds andconjugates.

Additional disclosure related to conjugates, linkers and methods forproducing conjugates is found in U.S. Application Publication No.2015/0157736, filed Nov. 26, 2014, the disclosure of which isincorporated herein by reference.

Target Polypeptides

Any of a wide variety of polypeptides can be modified to be conjugatedto a moiety of interest as described above. Polypeptides suitable formodification include both proteins having a naturally-occurring aminoacid sequence, fragments of naturally-occurring polypeptides, andnon-naturally occurring polypeptides and fragments thereof.

The following are examples of classes and types of polypeptides whichare of interest for modification using the compounds and methodsdescribed herein to produce the polypeptide conjugates described herein.

Therapeutic Polypeptides

In certain embodiments, the methods of producing a conjugate are appliedto modification of polypeptides that may provide for a therapeuticbenefit, such as those polypeptides for which attachment to a moiety canprovide for one or more of, for example, an increase in serum half-life,a decrease in an adverse immune response, additional or alternatebiological activity or functionality, and the like, or other benefit orreduction of an adverse side effect. Where the therapeutic polypeptideis an antigen for a vaccine, modification can provide for an enhancedimmunogenicity of the polypeptide.

Examples of classes of therapeutic proteins include those that arecytokines, chemokines, growth factors, hormones, antibodies, andantigens. Further examples include, but are not limited to, thefollowing: erythropoietin (EPO, e.g., native EPO or synthetic EPO (see,e.g., US 2003/0191291), such as, but not limited to, e.g., PROCRIT®,EPREX®, or EPOGEN® (epoetin-α), ARANESP® (darbepoietin-α), NEORECORMON®,EPOGIN® (epoetin-β), and the like); a growth hormone (e.g., asomatotropin, e.g., GENOTROPIN®, NUTROPIN®, NORDITROPIN®, SAIZEN®,SEROSTIM®, HUMATROPE®, etc.); human growth hormone (hGH); bovine growthhormone (bGH); follicle stimulating hormone (FSH); interferon (e.g.,IFN-γ, IFN-α, IFN-β, IFN-ω; IFN-τ, consensus interferon, and the like);insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.),insulin-like growth factor (e.g., IGF-I, IGF-II); blood factors (e.g.,Factor X, tissue plasminogen activator (TPA), and the like, such as, butnot limited to, e.g., ACTIVASE® (alteplase) tissue plasminogenactivator, NOVOSEVEN® (recombinant human factor VIIa), Factor VIIa,Factor VIII (e.g., KOGENATE®), Factor IX, 3-globin, hemoglobin, and thelike); colony stimulating factors (e.g., granulocyte-CSF (G-CSF, e.g.,NEUPOGEN® (filgrastim)), macrophage-CSF (M-CSF),granulocyte-macrophage-CSF (GM-CSF), Neulasta (pegfilgrastim),granulocyte-monocyte colony stimulating factor, megakaryocyte colonystimulating factor, and the like), transforming growth factors (e.g.,TGF-beta, TGF-alpha); interleukins (e.g., IL-1, IL-2 (e.g., Proleukin®),IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, and the like); a growthfactor (e.g., epidermal growth factor (EGF), platelet-derived growthfactor (PDGF, e.g., REGRANEX® (beclapermin)), fibroblast growth factors(FGFs, e.g., aFGF, bFGF, such as FIBLAST® (trafermin)), glial cellline-derived growth factor (GDNF), nerve growth factor (NGF), stem cellfactor (e.g., STEMGEN® (ancestim)), keratinocyte growth factor, ahepatocyte growth factor, and the like); a soluble receptor (e.g., aTNF-α-binding soluble receptor such as ENBREL® (etanercept), a solubleVEGF receptor, a soluble interleukin receptor, a soluble γ/δ T cellreceptor, and the like); an enzyme (e.g., α-glucosidase, CERAZYME®(imiglucarase, β-glucocerebrosidase, CEREDASE® (alglucerase); an enzymeactivator (e.g., tissue plasminogen activator); a chemokine (e.g.,IP-10, Mig, Groα/IL-8, regulated and normal T cell expressed andsecreted (RANTES), MIP-1α, MIP-1β, MCP-1, PF-4, and the like); anangiogenic agent (e.g., vascular endothelial growth factor (VEGF); ananti-angiogenic agent (e.g., a soluble VEGF receptor); a proteinvaccine; a neuroactive peptide such as bradykinin, cholecystokinin,gastin, secretin, oxytocin, gonadotropin-releasing hormone,beta-endorphin, enkephalin, substance P, somatostatin, galanin, growthhormone-releasing hormone, bombesin, warfarin, dynorphin, neurotensin,motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin,insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasinghormone, vasoactive intestinal peptide, a sleep peptide, etc.; otherproteins such as a thrombolytic agent, an atrial natriuretic peptide,bone morphogenic protein, thrombopoietin, relaxin, glial fibrillaryacidic protein, follicle stimulating hormone, a human alpha-1antitrypsin, a leukemia inhibitory factor, a transforming growth factor,a tissue factor, an insulin-like growth factor, a luteinizing hormone, afollicle stimulating hormone, a macrophage activating factor, tumornecrosis factor, a neutrophil chemotactic factor, a nerve growth factor,a tissue inhibitor of metalloproteinases; a vasoactive intestinalpeptide, angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitoryfactor; an IL-1 receptor antagonist (e.g., Kineret® (anakinra)); and thelike. It will be readily appreciated that native forms of the abovetherapeutic proteins are also of interest as target polypeptides in thepresent disclosure.

Further examples include antibodies, e.g., polyclonal antibodies,monoclonal antibodies, humanized antibodies, antigen-binding fragments(e.g., F(ab)′, Fab, Fv), single chain antibodies, and the like (e.g.,RITUXAN® (rituximab); REMICADE® (infliximab); HERCEPTIN® (trastuzumab);HUMIRA™ (adalimumab); XOLAIR® (omalizumab); BEXXAR® (tositumomab);RAPTIVA™ (efalizumab); ERBITUX™ (cetuximab); and the like). In someinstances, antibodies include antibodies that specifically bind to atumor antigen, an immune cell antigen (e.g., CD4, CD8, and the like), anantigen of a microorganism, particularly a pathogenic microorganism(e.g., a bacterial, viral, fungal, or parasitic antigen), and the like.

In some instances, the methods, conjugates and compounds describedherein can be applied to provide for a moiety (e.g., a water-solublepolymer) at a native or engineered site of glycosylation, such as foundin hyperglycosylated forms of a therapeutic protein.

The biological activity of a modified target polypeptide can be assayedaccording to methods known in the art. Modified polypeptides that retainat least one desired pharmacologic activity of the corresponding parentprotein are of interest.

Immunogenic Compositions

The methods, conjugates and compounds disclosed herein also findapplication in production of components of immunogenic compositions(e.g., therapeutic vaccines). For example, the compounds can be used tofacilitate attachment of moieties that increase serum half-life of apolypeptide antigen, that increase immunogenicity of the polypeptide, orthat link a non-amino acid antigen to a polypeptide carrier. In thisregard, the compounds can be used to facilitate modification ofmicrobial antigens (e.g., a bacterial, viral, fungal, or parasiticantigen), tumor antigens, and other antigens which are of interest foradministration to a subject to elicit an immune response in the subject.Also of interest is modification of antigens that are useful ineliciting antibodies which can be useful as research tools.

Further examples of polypeptides of interest for modification using thecompounds disclosed herein include those that are of interest fordetection or functional monitoring in an assay (e.g., as a researchtool, in a drug screening assay, and the like). Examples of polypeptidesof this type include receptors (e.g., G-protein coupled receptors(GPCRs, including orphan GPCRs)), receptor ligands (includingnaturally-occurring and synthetic), protein channels (e.g., ion channels(e.g., potassium channels, calcium channels, sodium channels, and thelike), and other polypeptides. In some embodiments, modification of cellsurface-associated polypeptides, such as transmembrane polypeptides) isof interest, for example where such modification is accomplished whilethe polypeptide is present in a membrane. Methods for modification of apolypeptide under physiological conditions are described further below.

Methods of Polypeptide Production

In general, the polypeptides described herein may be expressed inprokaryotes or eukaryotes in accordance with conventional ways,depending upon the purpose for expression. Thus, the present inventionfurther provides a host cell, e.g., a genetically modified host cellthat comprises a nucleic acid encoding a polypeptide.

Host cells for production (including large scale production) of anunconjugated or modified polypeptide suitable to form a conjugate asdescribed herein can be selected from any of a variety of available hostcells. Examples of host cells include those of a prokaryotic oreukaryotic unicellular organism, such as bacteria (e.g., Escherichiacoli strains, Bacillus spp. (e.g., B. subtilis), and the like) yeast orfungi (e.g., S. cerevisiae, Pichia spp., and the like), and other suchhost cells can be used. Examples of host cells originally derived from ahigher organism such as insects, vertebrates, including mammals, (e.g.,CHO, HEK, and the like), may be used as the expression host cells.

Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618 and CRL9096), CHO DG44 cells (Urlaub(1983) Cell 33:405), CHO—K1 cells (ATCC CCL-61), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems.

The expressed polypeptide can be recovered by any appropriate meansknown in the art. Further, any convenient protein purificationprocedures may be employed, where suitable protein purificationmethodologies are described in Guide to Protein Purification, (Deuthsered.) (Academic Press, 1990). For example, a lysate may prepared from acell comprising the expression vector expressing the desiredpolypeptide, and purified using high performance liquid chromatography(HPLC), exclusion chromatography, gel electrophoresis, affinitychromatography, and the like.

Methods for Modification of a Polypeptide

In certain embodiments, the polypeptide may be conjugated to a moiety ofinterest without first modifying the polypeptide. For instance, thepolypeptide may include one or more reactive groups suitable forconjugation to the moiety of interest (e.g., a moiety comprising acoupling moiety, such as a hydrazinyl-substituted heteroaryl compound orderivative thereof as described herein). In other embodiments, thepolypeptide may be modified before conjugation to the moiety ofinterest. Modification of the polypeptide may produce a modifiedpolypeptide that contains one or more reactive groups suitable forconjugation to the moiety of interest.

In some cases, the polypeptide may be modified at one or more amino acidresidues to provide one or more reactive groups suitable for conjugationto the moiety of interest (e.g., a moiety comprising a coupling moiety,such as a hydrazinyl-substituted heteroaryl compound or derivativethereof as described herein). For example, carbonyls introduced into apolypeptide can be selectively reacted with α-nucleophiles, such asaminooxy- and hydrazide-bearing compounds. Chemistries selective forcarbonyl functional groups on a protein with enhanced kinetics, siteselectivity and conjugate stability may result in improved bioconjugatesand provide access to new products and therapeutic targets as disclosedherein.

In certain embodiments, the polypeptide may be modified to include areactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehydemay be included in an “aldehyde tag” or “ald-tag”, which is meant torefer to an amino acid sequence derived from a sulfatase motif that hasbeen converted by action of a formylglycine generating enzyme (FGE) tocontain a 2-formylglycine residue (referred to herein as “fGly”). ThefGly residue generated by an FGE is also referred to in the literatureas a “formylglycine”. Stated differently, the term “aldehyde tag” isused herein to refer to an amino acid sequence comprising a “converted”sulfatase motif (i.e., a sulfatase motif in which the cysteine or theserine residue has been converted to fGly by action of an FGE, e.g.,L(fGly)TPSR). A converted sulfatase motif may be derived from an aminoacid sequence comprising an “unconverted” sulfatase motif (i.e., asulfatase motif in which the cysteine or serine residues has not beenconverted to fGly by an FGE, but is capable of being converted, e.g., anunconverted sulfatase motif with the sequence: L(C/S)TPSR). By“conversion” as used in the context of action of a formylglycinegenerating enzyme (FGE) on a sulfatase motif refers to biochemicalmodification of a cysteine or serine residue in a sulfatase motif to aformylglycine (fGly) residue (e.g., Cys to fGly, or Ser to fGly).Additional aspects of aldehyde tags and uses thereof in site-specificprotein modification are described in U.S. Pat. Nos. 8,846,866;8,729,232; 8,349,910; 8,097,701; and 7,985,783, the disclosures of eachof which are incorporated herein by reference.

Conversion of a polypeptide to include fGly can be accomplished bycell-based (in vivo) or cell-free methods (in vitro). Similarly,modification of a polypeptide to produce a polypeptide suitable forconjugation (e.g., modification to produce a polypeptide containing areactive group suitable for conjugation) can be accomplished bycell-based (in vivo) or cell-free methods (in vitro).

Alternatively, isolated, unmodified polypeptides can be isolatedfollowing recombinant production in a host cell lacking a suitableenzyme or by synthetic production. The isolated polypeptide may then becontacted with a suitable enzyme (e.g., a formylglycine generatingenzyme; FGE) under conditions to provide for the desired modification ofthe polypeptide to include fGly. The polypeptide can be unfolded bymethods known in the art (e.g., using heat, adjustment of pH, chaotropicagents, (e.g., urea, and the like), organic solvents (e.g.,hydrocarbons: octane, benzene, chloroform), etc.) and the denaturedprotein contacted with a suitable enzyme. The modified polypeptide canthen be refolded under suitable conditions.

Additional aspects of suitable enzymes (e.g., FGEs) and uses thereof insite-specific protein modification are described in U.S. applicationSer. No. 14/975,403, filed Dec. 18, 2015, the disclosure of which isincorporated herein by reference.

In some cases, the modified polypeptide containing the fGly residue maybe conjugated to the moiety of interest by reaction of the fGly with acompound as described herein (e.g., a compound containing a couplingmoiety, such as a hydrazinyl-substituted heteroaryl compound orderivative thereof as described herein). For example, an fGly-containingpolypeptide may be isolated from a production source (e.g., recombinanthost cell production, synthetic production), and contacted with areactive partner-containing drug or other moiety (e.g., detectablelabel) under conditions suitable to provide for conjugation of the drugor other moiety to the polypeptide. For example, the reactivepartner-containing drug or other moiety may include a reactive moiety(e.g., a hydrazinyl-substituted heteroaryl compound or derivativethereof as described herein). The hydrazinyl-substitutedheteroaryl-containing drug or other moiety may be reacted with thepolypeptide to produce a polypeptide conjugate as described herein.

To conjugate a hydrazinyl-substituted heteroaryl-modified drug to analdehyde-tagged antibody as described herein, the following generalprotocol may be used. A desired amount of the hydrazinyl-substitutedheteroaryl-modified drug may be reacted with an appropriatealdehyde-tagged antibody (e.g., an aldehyde-tagged antibody (dimer) withone fGly (e.g., reactive aldehyde group) per chain). After the reactionis complete, unreacted drug can be removed using diafiltration. Mono-and di-conjugated species may then be purified away from unconjugatedmaterial, for example using hydrophobic interaction chromatography(HIC). The enriched sample may be put into a buffer (e.g., a finalformulation buffer, such as phosphate buffered saline (PBS)), forexample by using diafiltration. The final sample may be analyzed usinghydrophobic interaction chromatography (HIC) to determine the drug toantibody ratio (DAR) and/or may be analyzed using size exclusionchromatography (SEC) to determine the level of aggregation.

Polypeptide Conjugates

The polypeptides can be subjected to conjugation to provide forattachment of a wide variety of moieties. Examples of moieties ofinterest include, but are not limited to, a drug, a detectable label, asmall molecule, a water-soluble polymer, a peptide, and the like (alsoreferred to a “payload” or “cargo” herein). Thus, the present disclosureprovides a polypeptide conjugate as described above.

The moiety of interest is provided as a component of a reactive partnerfor reaction with a residue of a polypeptide. In certain embodiments,the methods of polypeptide conjugation are compatible with reactionconditions suitable for the polypeptide. For example, the reactionconditions may include a reaction mixture that includes water. In somecases, the reaction mixture may have a pH compatible with thepolypeptide, such as, but not limited to, a pH of 4 to 11, or a pH of 5to 10, or a pH of 6 to 9, or a pH of 6 to 8. In certain instances, thereaction mixture has a pH of 7. In some embodiments, the reactionconditions are performed at a temperature compatible with thepolypeptide. For example, the reaction conditions may be at atemperature of 20° C. to 45° C., such as 25° C. to 40° C., or 30 OC to40° C., or 35° C. to 40° C. In some cases, the reaction conditions areat room temperature (e.g., 25° C.). In some instances, the reactionconditions are at a temperature of 37° C.

Provided the present disclosure, the ordinarily skilled artisan canreadily adapt any of a variety of moieties to provide a reactive partnerfor conjugation to a polypeptide as contemplated herein. The ordinarilyskilled artisan will appreciate that factors such as pH and sterichindrance (i.e., the accessibility of the modified amino acid residue toreaction with a reactive partner of interest) are of importance.Modifying reaction conditions to provide for optimal conjugationconditions is well within the skill of the ordinary artisan, and isroutine in the art. Where conjugation is conducted with a polypeptidepresent in or on a living cell, the conditions are selected so as to bephysiologically compatible. For example, the pH can be droppedtemporarily for a time sufficient to allow for the reaction to occur butwithin a period tolerated by the cell (e.g., from about 30 min to 1hour). Physiological conditions for conducting modification ofpolypeptides on a cell surface can be similar to those used in aketone-azide reaction in modification of cells bearing cell-surfaceazides (see, e.g., U.S. Pat. No. 6,570,040).

In certain embodiments, the present disclosure provides a polypeptideconjugate, where the polypeptide is an antibody. As such, embodimentsinclude an antibody conjugated to a moiety of interest, where anantibody conjugated to a moiety of interest is referred to as an“antibody conjugate.” An Ig polypeptide generally includes at least anIg heavy chain constant region or an Ig light chain constant region, andcan further include an Ig variable region (e.g., a V_(L) region and/or aV_(H) region). Ig heavy chain constant regions include Ig constantregions of any heavy chain isotype, non-naturally occurring Ig heavychain constant regions (including consensus Ig heavy chain constantregions). An Ig constant region can be modified to be conjugated to amoiety of interest, where the moiety of interest is present in oradjacent a solvent-accessible loop region of the Ig constant region.

In some cases, an antibody conjugate of the present disclosure caninclude: 1) Ig heavy chain constant region conjugated to one or moremoieties of interest, and an Ig light chain constant region conjugatedto one or more moieties of interest; 2) an Ig heavy chain constantregion conjugated to one or more moieties of interest, and an Ig lightchain constant region that is not conjugated to a moiety of interest; or3) an Ig heavy chain constant region that is not conjugated to a moietyof interest, and an Ig light chain constant region conjugated to one ormore moieties of interest. A subject antibody conjugate can also includevariable VH and/or VL domains. As described above, the one or moremoieties of interest may be conjugated to the Ig heavy chain constantregion or the Ig light chain constant region at a single amino acidresidue (e.g., one or two moieties of interest conjugated to a singleamino acid residue), or conjugated to the Ig heavy chain constant regionand/or the Ig light chain constant region at two or more different aminoacid residues.

An antibody conjugate of the present disclosure can include, as theconjugated moiety, any of a variety of compounds, as described herein,e.g., a drug (e.g., a peptide drug, a small molecule drug, and thelike), a water-soluble polymer, a detectable label, a synthetic peptide,etc.

An antibody conjugate can have any of a variety of antigen-bindingspecificities, as described above, including, e.g., an antigen presenton a cancer cell; an antigen present on an autoimmune cell; an antigenpresent on a pathogenic microorganism; an antigen present on avirus-infected cell (e.g., a human immunodeficiency virus-infectedcell), e.g., CD4 or gp120; an antigen present on a diseased cell; andthe like. For example, an antibody conjugate can bind an antigen, asnoted above, where the antigen is present on the surface of the cell. Anantibody conjugate of the present disclosure can bind antigen with asuitable binding affinity, e.g., from 5×10⁻⁶ M to 10⁻⁷ M, from 10⁻⁷ M to5×10⁻⁷ M, from 5×10⁻⁷ M to 10⁻⁸ M, from 10⁻⁸ M to 5×10⁻⁸M, from 5×10⁻⁸ Mto 10⁻⁹ M, or a binding affinity greater than 10⁻⁹ M.

As non-limiting examples, a subject antibody conjugate can bind anantigen present on a cancer cell (e.g., a tumor-specific antigen; anantigen that is over-expressed on a cancer cell; etc.), and theconjugated moiety can be a cytotoxic compound (e.g., a cytotoxic smallmolecule, a cytotoxic synthetic peptide, etc.). For example, a subjectantibody conjugate can be specific for an antigen on a cancer cell,where the conjugated moiety is a cytotoxic compound (e.g., a cytotoxicsmall molecule, a cytotoxic synthetic peptide, etc.).

As further non-limiting examples, a subject antibody conjugate can bindan antigen present on a cell infected with a virus (e.g., where theantigen is encoded by the virus; where the antigen is expressed on acell type that is infected by a virus; etc.), and the conjugated moietycan be a viral fusion inhibitor. For example, a subject antibodyconjugate can bind an antigen present on a cell infected with a virus,and the conjugated moiety can be a viral fusion inhibitor.

Embodiments of the present disclosure also include polypeptideconjugates where the polypeptide is a carrier protein. For example,carrier proteins can be covalently and site-specifically bound to drugto provide a drug-containing scaffold. A carrier protein can besite-specifically conjugated to a covalently bound molecule of interest,such as a drug (e.g., a peptide, a small molecule drug, and the like),detectable label, etc. In certain embodiments, drug-scaffold conjugatescan provide for enhanced serum half-life of the drug.

In general a “carrier protein” is a protein that is biologically inert,is susceptible to modification as disclosed herein, and which canprovide for solvent-accessible presentation of the moiety of interestconjugated to the carrier protein through a modified amino acid residuein the carrier protein (e.g., through an oxime or hydrazone bond withinthe converted sulfatase motif of an aldehyde tagged carrier protein) ina physiological environment. “Biologically inert” is meant to indicatethe carrier protein exhibits clinically insignificant or no detectablebiological activity when administered to the appropriate subject, suchas when administered to a human subject. Thus, carrier proteins arebiologically inert in that they, for example, are of low immunogenicity,do not exhibit significant or detectable targeting properties (e.g., donot exhibit significant or detectable activity in binding to a specificreceptor), and exhibit little or no detectable biological activity thatmay interfere with activity of the moiety (e.g., drug or detectablelabel) conjugated to the aldehyde-tagged carrier protein. By “lowimmunogenicity” is meant that the carrier protein elicits little or nodetectable immune response upon administration to a subject, such as amammalian subject, e.g., a human subject. Carrier proteins can beprovided in monomeric or multimeric (e.g., dimeric) forms.

Carrier proteins having a three-dimensional structure when folded thatprovides for multiple different solvent-accessible sites that areamenable to modification (and thus conjugation to a moiety of interest)are of interest. In general, carrier proteins of interest are those thatare of a size and three-dimensional folded structure so as to providefor presentation of the conjugated moiety of interest on solventaccessible surfaces in a manner that is sufficiently spatially separatedso as to provide for activity and bioavailability of the conjugatedmoiety or moieties of interest. The carrier protein may be selectedaccording to a variety of factors including, but not limited to, themoiety (e.g., drug or detectable label) to be conjugated to the carrierprotein.

Accordingly, any of a wide variety of polypeptides can be suitable foruse as carrier proteins for use in the carrier protein conjugates of thepresent disclosure. Such carrier proteins can include those having anaturally-occurring amino acid sequence, fragments ofnaturally-occurring polypeptides, and non-naturally occurringpolypeptides and fragments thereof.

Examples of carrier proteins include, but are not limited to, albuminand fragments thereof (e.g., human serum albumin, bovine serum albumin,and the like), transferrin and fragments thereof (e.g. humantransferrin), and Fc fragments having reduced binding to a mammalian Fcreceptor, particularly a human Fc receptor (e.g., a modified Fc fragmentof an antibody (e.g., IgG), such as a mammalian antibody, e.g., a humanantibody). Examples of modified Fc fragments having reduced Fc receptorbinding are exemplified by the Fc fragments of Herceptin (trastuzumab)and Rituxan (Rituximab), which contain point mutations that provide forreduced Fc receptor binding (see, e.g., Clynes et al., Nature Medicine(2000), 6, 443-446). Alternatively or in addition, the isotype of the Fcfragment can be selected according to a desired level of Fc receptorbinding (e.g., use of an Fc fragment of an IgG4 isotype human heavychain constant region rather than from IgG1 or IgG3. (see, e.g., FridmanFASEB J 1991 September; 5 (12): 2684-90). In general, carrier proteinscan be at least about 4 kDa (e.g., about 50 amino acid residues inlength), usually at least about 25 kDa, and can be larger in size (e.g.,transferrin has a molecular weight of 90 kDa while Fc fragments can havemolecular weights of 30 kDa to 50 kDa).

The conjugates described herein can be used for a variety ofapplications including, but not limited to, visualization usingfluorescence or epitope labeling (e.g., electron microscopy using goldparticles equipped with reactive groups for conjugation to the compoundsand conjugates described herein); protein immobilization (e.g., proteinmicroarray production); protein dynamics and localization studies andapplications; and conjugation of proteins with a moiety of interest(e.g., moieties that improve a parent protein's half-life (e.g.,poly(ethylene glycol)), targeting moieties (e.g., to enhance delivery toa site of action), and biologically active moieties (e.g., a therapeuticmoiety).

The polypeptide conjugate may include a polypeptide conjugated to amoiety or moieties that provide for one or more of a wide variety offunctions or features. In general, examples of moieties include, but arenot limited to, the following: detectable labels (e.g., fluorescentlabels); light-activated dynamic moieties (e.g., azobenzene mediatedpore closing, azobenzene mediated structural changes, photodecagingrecognition motifs); water soluble polymers (e.g., PEGylation);purification tags (e.g., to facilitate isolation by affinitychromatography (e.g., attachment of a FLAG epitope); membranelocalization domains (e.g., lipids or glycophosphatidylinositol(GPI)-type anchors); immobilization tags (e.g., to facilitate attachmentof the polypeptide to a surface, including selective attachment); drugs(e.g., to facilitate drug targeting, e.g., through attachment of thedrug to an antibody); targeted delivery moieties, (e.g., ligands forbinding to a target receptor (e.g., to facilitate viral attachment,attachment of a targeting protein present on a liposome, etc.)), and thelike.

Specific, non-limiting examples are provided below.

Drugs for Conjugation to a Polypeptide

Any of a number of drugs are suitable for use, or can be modified to berendered suitable for use, as a reactive partner to conjugate to apolypeptide. Examples of drugs include small molecule drugs and peptidedrugs. Thus, the present disclosure provides drug-polypeptideconjugates.

“Small molecule drug” as used herein refers to a compound, e.g., anorganic compound, which exhibits a pharmaceutical activity of interestand which is generally of a molecular weight of 800 Da or less, or 2000Da or less, but can encompass molecules of up to 5 kDa and can be aslarge as 10 kDa. A small inorganic molecule refers to a moleculecontaining no carbon atoms, while a small organic molecule refers to acompound containing at least one carbon atom.

“Peptide drug” as used herein refers to amino-acid containing polymericcompounds, and is meant to encompass naturally-occurring andnon-naturally-occurring peptides, oligopeptides, cyclic peptides,polypeptides, and proteins, as well as peptide mimetics. The peptidedrugs may be obtained by chemical synthesis or be produced from agenetically encoded source (e.g., recombinant source). Peptide drugs canrange in molecular weight, and can be from 200 Da to 10 kDa or greaterin molecular weight.

In some cases, the drug is a cancer chemotherapeutic agent. For example,where the polypeptide is an antibody (or fragment thereof) that hasspecificity for a tumor cell, the antibody can be modified as describedherein to include a modified amino acid, which can be subsequentlyconjugated to a cancer chemotherapeutic agent. Cancer chemotherapeuticagents include non-peptidic (i.e., non-proteinaceous) compounds thatreduce proliferation of cancer cells, and encompass cytotoxic agents andcytostatic agents. Non-limiting examples of chemotherapeutic agentsinclude alkylating agents, nitrosoureas, antimetabolites, antitumorantibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidiccompounds can also be used.

Suitable cancer chemotherapeutic agents include dolastatin and activeanalogs and derivatives thereof; and auristatin and active analogs andderivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethylauristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See,e.g., WO 96/33212, WO 96/14856, and U.S. Pat. No. 6,323,315. Forexample, dolastatin 10 or auristatin PE can be included in anantibody-drug conjugate of the present disclosure. Suitable cancerchemotherapeutic agents also include maytansinoids and active analogsand derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996)Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and activeanalogs and derivatives thereof (e.g., including the syntheticanalogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogsand derivatives thereof (e.g., pyrrolobenzodiazepine (PBD).

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel(Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicimmunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf),rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®.Estrogens stimulate proliferation and differentiation; thereforecompounds that bind to the estrogen receptor are used to block thisactivity. Corticosteroids may inhibit T cell proliferation.

Other suitable chemotherapeutic agents include metal complexes, e.g.cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; andhydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomeraseinhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

Taxanes are suitable for use. “Taxanes” include paclitaxel, as well asany active taxane derivative or pro-drug. “Paclitaxel” (which should beunderstood herein to include analogues, formulations, and derivativessuch as, for example, docetaxel, TAXOL™ TAXOTERE™ (a formulation ofdocetaxel), 10-desacetyl analogs of paclitaxel and3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs of paclitaxel) may bereadily prepared utilizing techniques known to those skilled in the art(see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949;5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtainedfrom a variety of commercial sources, including for example, SigmaChemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Biological response modifiers suitable for use include, but are notlimited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2)inhibitors of serine/threonine kinase activity; (3) tumor-associatedantigen antagonists, such as antibodies that bind specifically to atumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6)IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) inhibitors ofangiogenesis.

Examples of drugs include small molecule drugs, such as a cancerchemotherapeutic agent. For example, where the polypeptide is anantibody (or fragment thereof) that has specificity for a tumor cell,the antibody can be modified as described herein to include a modifiedamino acid, which can be subsequently conjugated to a cancerchemotherapeutic agent, such as a microtubule affecting agent. Incertain embodiments, the drug is a microtubule affecting agent that hasantiproliferative activity, such as a maytansinoid. In certainembodiments, the drug is a maytansinoid, which as the followingstructure:

where

indicates the point of attachment between the maytansinoid and thelinker, L, in conjugates and compounds described herein. By “point ofattachment” is meant that the

symbol indicates the bond between the N of the maytansinoid and thelinker, L, in conjugates and compounds described herein. For example, informula (I), W may be a maytansinoid, such as a maytansinoid of thestructure above, where

indicates the point of attachment between the maytansinoid and thelinker, L. In some cases, the maytansinoid of the structure above may bereferred to as a deacyl maytansine.

Methods for Modification of Drugs to Contain a Reactive Partner

Drugs to be conjugated to a polypeptide may be modified to incorporate areactive partner for reaction with the polypeptide. Where the drug is apeptide drug, the reactive moiety (e.g., aminooxy or hydrazide can bepositioned at an N-terminal region, the N-terminus, a C-terminal region,the C-terminus, or at a position internal to the peptide. For example,an example of a method involves synthesizing a peptide drug having anaminooxy group. In this example, the peptide is synthesized from aBoc-protected precursor. An amino group of a peptide can react with acompound comprising a carboxylic acid group and oxy-N-Boc group. As anexample, the amino group of the peptide reacts with3-(2,5-dioxopyrrolidin-1-yloxy)propanoic acid. Other variations on thecompound comprising a carboxylic acid group and oxy-N-protecting groupcan include different number of carbons in the alkylene linker andsubstituents on the alkylene linker. The reaction between the aminogroup of the peptide and the compound comprising a carboxylic acid groupand oxy-N-protecting group occurs through standard peptide couplingchemistry. Examples of peptide coupling reagents that can be usedinclude, but not limited to, DCC (dicyclohexylcarbodiimide), DIC(diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP(1-benzotriazolediethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC(1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuricfluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidiniumhexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP(benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate), HBTU(O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate),TBTU (O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumtetrafluoroborate), TSTU(O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate),HATU(N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide), BOP-Cl(bis(2-oxo-3-oxazolidinyl)phosphinic chloride), PyBOP((1-H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrolidino)phosphoniumtetrafluorophopsphate), BrOP (bromotris(dimethylamino)phosphoniumhexafluorophosphate), DEPBT(3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) PyBrOP(bromotris(pyrrolidino)phosphonium hexafluorophosphate). As anon-limiting example, HOBt and DIC can be used as peptide couplingreagents.

Deprotection to expose the amino-oxy functionality is performed on thepeptide comprising an N-protecting group. Deprotection of theN-oxysuccinimide group, for example, occurs according to standarddeprotection conditions for a cyclic amide group. Deprotectingconditions can be found in Greene and Wuts, Protective Groups in OrganicChemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al.Certain deprotection conditions include a hydrazine reagent, aminoreagent, or sodium borohydride. Deprotection of a Boc protecting groupcan occur with TFA. Other reagents for deprotection include, but are notlimited to, hydrazine, methylhydrazine, phenylhydrazine, sodiumborohydride, and methylamine. The product and intermediates can bepurified by conventional means, such as HPLC purification.

The ordinarily skilled artisan will appreciate that factors such as pHand steric hindrance (i.e., the accessibility of the amino acid residueto reaction with a reactive partner of interest) are of importance,Modifying reaction conditions to provide for optimal conjugationconditions is well within the skill of the ordinary artisan, and isroutine in the art. Where conjugation is conducted with a polypeptidepresent in or on a living cell, the conditions are selected so as to bephysiologically compatible. For example, the pH can be droppedtemporarily for a time sufficient to allow for the reaction to occur butwithin a period tolerated by the cell (e.g., from about 30 min to 1hour). Physiological conditions for conducting modification ofpolypeptides on a cell surface can be similar to those used in aketone-azide reaction in modification of cells bearing cell-surfaceazides (see, e.g., U.S. Pat. No. 6,570,040).

Small molecule compounds containing, or modified to contain, anα-nucleophilic group that serves as a reactive partner with a compoundor conjugate disclosed herein are also contemplated for use as drugs inthe polypeptide-drug conjugates of the present disclosure. Generalmethods are known in the art for chemical synthetic schemes andconditions useful for synthesizing a compound of interest (see, e.g.,Smith and March, March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; orVogel, A Textbook of Practical Organic Chemistry, Including QualitativeOrganic Analysis, Fourth Edition, New York: Longman, 1978).

Peptide Drugs

In some cases, a conjugate comprises a covalently linked peptide.Suitable peptides include, but are not limited to, cytotoxic peptides;angiogenic peptides; anti-angiogenic peptides; peptides that activate Bcells; peptides that activate T cells; anti-viral peptides; peptidesthat inhibit viral fusion; peptides that increase production of one ormore lymphocyte populations; anti-microbial peptides; growth factors;growth hormone-releasing factors; vasoactive peptides; anti-inflammatorypeptides; peptides that regulate glucose metabolism; an anti-thromboticpeptide; an anti-nociceptive peptide; a vasodilator peptide; a plateletaggregation inhibitor; an analgesic; and the like.

In some embodiments, the peptide can be chemically synthesized toinclude a group reactive with an amino acid residue or a modified aminoacid residue of the polypeptide. A suitable synthetic peptide has alength of from 5 amino acids to 100 amino acids, or longer than 100amino acids; e.g., a suitable peptide has a length of from 5 amino acids(aa) to 10 aa, from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, from 40 aa to 50 aa,from 50 aa to 60 aa, from 60 aa to 70 aa, from 70 aa to 80 aa, from 80aa to 90 aa, or from 90 aa to 100 aa.

In certain embodiments, a peptide can be modified to contain anα-nucleophile-containing moiety (e.g., an aminooxy or hydrazide moiety),e.g., can be reacted with an fGly-containing polypeptide to yield aconjugate in which the polypeptide and peptide are linked by a hydrazoneor oxime bond, respectively. Examples of methods of synthesizing apeptide, such that the synthetic peptide comprising a reactive groupreactive with an amino acid residue or a modified amino acid residue ofthe polypeptide, are described above.

Suitable peptides include, but are not limited to, hLF-11 (an 11-aminoacid N-terminal fragment of lactoferrin), an anti-microbial peptide;granulysin, an anti-microbial peptide; Plectasin (NZ2114; SAR 215500),an anti-microbial peptide; viral fusion inhibitors such as Fuzeon(enfuvirtide), TRI-1249 (T-1249; see, e.g., Matos et al. (2010) PLoS One5:e9830), TRI-2635 (T-2635; see, e.g., Eggink et al. (2009) J. Biol.Chem. 284:26941), T651, and TRI-1144; C5a receptor inhibitors such asPMX-53, JPE-1375, and JSM-7717; POT-4, a human complement factor C3inhibitor; Pancreate (an INGAP derivative sequence, a HIP-human proisletprotein); somatostatin; a somatostatin analog such as DEBIO 8609(Sanvar), octreotide, octreotide (C2L), octreotide QLT, octreotide LAR,Sandostatin LAR, SomaLAR, Somatuline (lanreotide), see, e.g., Deghenghiet al. (2001) Endocrine 14:29; TH9507 (Tesamorelin, a growthhormone-releasing factor); POL7080 (a protegrin analog, ananti-microbial peptide); relaxin; a corticotropin releasing factoragonist such as urotensin, sauvagine, and the like; a heat shock proteinderivative such as DiaPep277; a human immunodeficiency virus entryinhibitor; a heat shock protein-20 mimic such as AZX100; a thrombinreceptor activating peptide such as TP508 (Chrysalin); a urocortin 2mimic (e.g., a CRF2 agonist) such as urocortin-2; an immune activatorsuch as Zadaxin (thymalfasin; thymosin-α1), see, e.g., Sjogren (2004) J.Gastroenterol. Hepatol. 19:S69; a hepatitis C virus (HCV) entryinhibitorE2 peptide such as HCV3; an atrial natriuretic peptide such asHANP (Sun 4936; carperitide); an annexin peptide; a defensin(anti-microbial peptide) such as hBD2-4; a defensin (anti-microbialpeptide) such as hBD-3; a defensin (anti-microbial peptide) such asPMX-30063; a histatin (anti-microbial peptide) such as histatin-3,histatin-5, histatin-6, and histatin-9; a histatin (anti-microbialpeptide) such as PAC-113; an indolicidin (anti-microbial peptide) suchas MX-594AN (Omniganin; CLS001); an indolicidin (anti-microbial peptide)such as Omnigard (MBI-226; CPI-226); an anti-microbial peptide such asan insect cecropin; an anti-microbial peptide such as a lactoferrin(talactoferrin); an LL-37/cathelicidin derivative (an anti-microbialpeptide) such as P60.4 (OP-145); a magainin (an anti-microbial peptide)such as Pexiganan (MSI-78; Suponex); a protegrin (an anti-microbialpeptide) such as IB-367 (Iseganan); an agan peptide; a beta-natriureticpeptide such as Natrecor, or Noratak (Nesiritide), or ularitide;bivalarudin (Angiomax), a thrombin inhibitor; a C peptide derivative; acalcitonin such as Miacalcin (Fortical); an enkephalin derivative; anerythropoiesis-stimulating peptide such as Hematide; a gap junctionmodulator such as Danegaptide (ZP1609); a gastrin-releasing peptide; aghrelin; a glucagon-like peptide; a glucagon-like peptide-2 analog suchas ZP1846 or ZP1848; a glucosaminyl muramyl dipeptide such as GMDP; aglycopeptide antibiotic such as Oritavancin; a teicoplanin derivativesuch as Dalbavancin; a gonadotropin releasing hormone (GnRH) such asZoladex (Lupon) or Triptorelin; a histone deacetylase (HDAC) inhibitordepsipeptide such as PM02734 (Irvalec); an integrin such aseptifibatide; an insulin analog such as Humulog; a kahalalidedepsipeptide such as PM02734; a kallikrein inhibitor such as Kalbitor(ecallantide); an antibiotic such as Telavancin; a lipopeptide such asCubicin or MX-2401; a lutenizing hormone releasing hormone (LHRH) suchas goserelin; an LHRH synthetic decapeptide agonist analog such asTreistar (triptorelin pamoate); an LHRH such as Eligard; an M2 proteinchannel peptide inhibitor; metreleptin; a melanocortin receptor agonistpeptide such as bremalanotide/PT-141; a melanocortin; a muramyltripeptide such as Mepact (mifamurtide); a myelin basic protein peptidesuch as MBP 8298 (dirucotide); an N-type voltage-gated calcium channelblocker such as Ziconotide (Prialt); a parathyroid hormone peptide; aparathyroid analog such as 768974; a peptide hormone analog such asUGP281; a prostaglandin F2-α receptor inhibitor such as PDC31; aprotease inhibitor such as PPL-100; surfaxin; a thromobspondin-1 (TSP-1)mimetic such as CVX-045 or ABT 510; a vasoactive intestinal peptide;vasopressin; a Y2R agonist peptide such as RG7089; obinepeptide; andTM30339.

Detectable Labels

The conjugates, compounds and methods of the present disclosure can beused to conjugate a detectable label to polypeptide. Examples ofdetectable labels include, but are not limited to, fluorescent molecules(e.g., autofluorescent molecules, molecules that fluoresce upon contactwith a reagent, etc.), radioactive labels (e.g., ¹¹¹In, ¹²⁵I, ¹³¹I,²¹²B, ⁹⁰Y, ¹⁸⁶Rh, and the like), biotin (e.g., to be detected throughreaction of biotin and avidin), fluorescent tags, imaging reagents, andthe like. Detectable labels also include peptides or polypeptides thatcan be detected by antibody binding, e.g., by binding of a detectablylabeled antibody or by detection of bound antibody through asandwich-type assay. Further examples of detectable labels include, butare not limited to, dye labels (e.g., chromophores, fluorophores, suchas, but not limited to, Alexa Fluor® fluorescent dyes (e.g., AlexaFluor® 350, 405, 430, 488, 532, 546, 555, 568, 594, 595, 610, 633, 635,647, 660, 680, 700, 750, 790, and the like), coumarins, rhodamines(5-carboxyrhodamine and sulfo derivates thereof, e.g.,5-carboxy-disulfo-rhodamine, carbopyranins and oxazines, such as ATTOdyes (e.g., ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594,610, 611X, 620, 633, 635, 637, 647, 647N, 655, 665, 680, 700, 725 or740), biophysical probes (spin labels, nuclear magnetic resonance (NMR)probes), Förster Resonance Energy Transfer (FRET)-type labels (e.g., atleast one member of a FRET pair, including at least one member of afluorophore/quencher pair), Bioluminescence Resonance Energy Transfer(BRET)-type labels (e.g., at least one member of a BRET pair),immunodetectable tags (e.g., FLAG, His(6), and the like), localizationtags (e.g., to identify association of a tagged polypeptide at thetissue or molecular cell level (e.g., association with a tissue type, orparticular cell membrane), and the like.

Attachment of Moieties for Delivery to a Target Site

Embodiments of the present disclosure also include a polypeptideconjugated to one or more moieties, such as, but not limited to, a drug(e.g., a small molecule drug), toxin, or other molecule for delivery toa target site (e.g., a cell) and which can provide for a pharmacologicalactivity or can serve as a target for delivery of other molecules.

Also contemplated are conjugates that include one of a pair of bindingpartners (e.g., a ligand, a ligand-binding portion of a receptor, areceptor-binding portion of a ligand, etc.). For example, the conjugatecan include a polypeptide that serves as a viral receptor and, uponbinding with a viral envelope protein or viral capsid protein,facilitates attachment of virus to the cell surface on which themodified polypeptide is expressed. Alternatively, the conjugate mayinclude an antigen that is specifically bound by an antibody (e.g.,monoclonal antibody), to facilitate detection and/or separation of hostcells expressing the modified polypeptide.

Attachment of Target Molecules to a Support

The methods can provide for conjugation of a polypeptide to a moiety tofacilitate attachment of the polypeptide to a solid substrate (e.g., tofacilitate assays), or to a moiety to facilitate easy separation (e.g.,a hapten recognized by an antibody bound to a magnetic bead). In someembodiments, the methods are used to provide for attachment of a proteinto an array (e.g., chip) in a defined orientation. For example, apolypeptide modified at a selected site (e.g., at or near theN-terminus) can be generated, and the methods, conjugates and compoundsused to deliver a moiety to the modified polypeptide. The moiety canthen be used as the attachment site for affixing the polypeptide to asupport (e.g., solid or semi-solid support, such as a support suitablefor use as a microchip in high-throughput assays).

Water-Soluble Polymers

In some cases, a conjugate includes a covalently linked water-solublepolymer. A moiety of particular interest is a water-soluble polymer. A“water-soluble polymer” refers to a polymer that is soluble in water andis usually substantially non-immunogenic, and usually has an atomicmolecular weight greater than 1,000 Daltons. The methods, conjugates andcompounds described herein can be used to attach one or morewater-soluble polymers to a polypeptide. Attachment of a water-solublepolymer (e.g., PEG) to a polypeptide, such as a pharmaceutically active(e.g., therapeutic) polypeptide can be desirable as such modificationcan increase the therapeutic index by increasing serum half-life as aresult of increased proteolytic stability and/or decreased renalclearance. Additionally, attachment of one or more polymers (e.g.,PEGylation) can reduce immunogenicity of protein pharmaceuticals.

In some embodiments, the water-soluble polymer has an effectivehydrodynamic molecular weight of greater than 5,000 Da, greater than10,000 Da, greater than 20,000 to 500,000 Da, greater than 40,000 Da to300,000 Da, greater than 50,000 Da to 70,000 Da, such as greater than60,000 Da. In some embodiments, the water-soluble polymer has aneffective hydrodynamic molecular weight of from 10 kDa to 20 kDa, from20 kDa to 25 kDa, from 25 kDa to 30 kDa, from 30 kDa to 50 kDa, or from50 kDa to 100 kDa. By “effective hydrodynamic molecular weight” isintended the effective water-solvated size of a polymer chain asdetermined by aqueous-based size exclusion chromatography (SEC). Whenthe water-soluble polymer contains polymer chains having polyalkyleneoxide repeat units, such as ethylene oxide repeat units, each chain canhave an atomic molecular weight of 200 Da to 80,000 Da, or 1,500 Da to42,000 Da, including 2,000 to 20,000 Da. Unless referred tospecifically, molecular weight is intended to refer to atomic molecularweight. Linear, branched, and terminally charged water soluble polymers(e.g., PEG) may be used.

Polymers useful as moieties to be attached to a polypeptide can have awide range of molecular weights, and polymer subunits. These subunitsmay include a biological polymer, a synthetic polymer, or a combinationthereof. Examples of such water-soluble polymers include: dextran anddextran derivatives, including dextran sulfate, P-amino cross linkeddextrin, and carboxymethyl dextrin, cellulose and cellulose derivatives,including methylcellulose and carboxymethyl cellulose, starch anddextrines, and derivatives and hydroylactes of starch, polyalkyleneglycol and derivatives thereof, including polyethylene glycol,methoxypolyethylene glycol, polyethylene glycol homopolymers,polypropylene glycol homopolymers, copolymers of ethylene glycol withpropylene glycol, wherein said homopolymers and copolymers areunsubstituted or substituted at one end with an alkyl group, heparin andfragments of heparin, polyvinyl alcohol and polyvinyl ethyl ethers,polyvinylpyrrolidone, aspartamide, and polyoxyethylated polyols, withthe dextran and dextran derivatives, dextrine and dextrine derivatives.It will be appreciated that various derivatives of the specificallyrecited water-soluble polymers are also contemplated.

Water-soluble polymers such as those described above includepolyalkylene oxide based polymers, such as polyethylene glycol “PEG”(See. e.g., “Poly(ethylene glycol) Chemistry: Biotechnical andBiomedical Applications”, J. M. Harris, Ed., Plenum Press, New York,N.Y. (1992); and “Poly(ethylene glycol) Chemistry and BiologicalApplications”, J. M. Harris and S. Zalipsky, Eds., ACS (1997); andInternational Patent Applications: WO 90/13540, WO 92/00748, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28937, WO 95/11924, WO 96/00080, WO 96/23794, WO 98/07713, WO98/41562, WO 98/48837, WO 99/30727, WO 99/32134, WO 99/33483, WO99/53951, WO 01/26692, WO 95/13312, WO 96/21469, WO 97/03106, WO99/45964, and U.S. Pat. Nos. 4,179,337; 5,075,046; 5,089,261; 5,100,992;5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838;5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001;5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805;5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662;5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990;5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900;5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455;5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042;6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095;6,194,580; 6,214,966).

Examples of polymers of interest include those containing a polyalkyleneoxide, polyamide alkylene oxide, or derivatives thereof, includingpolyalkylene oxide and polyamide alkylene oxide comprising an ethyleneoxide repeat unit of the formula —(CH₂—CH₂—O)—. Further examples ofpolymers of interest include a polyamide having a molecular weightgreater than 1,000 Daltons of the formula —[C(O)—X—C(O)—NH—Y—NH]n- or—[NH—Y—NH—C(O)—X—C(O)]_(n)—, where X and Y are divalent radicals thatmay be the same or different and may be branched or linear, and n is adiscrete integer from 2-100, such as from 2 to 50, and where either orboth of X and Y comprises a biocompatible, substantially non-antigenicwater-soluble repeat unit that may be linear or branched. Furtherexamples of water-soluble repeat units comprise an ethylene oxide of theformula —(CH₂—CH₂—O)— or —(O—CH₂—CH₂)—. The number of such water-solublerepeat units can vary significantly, with the number of such units beingfrom 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, for example from2 to 50. An example of an embodiment is one in which one or both of Xand Y is selected from: —((CH₂)_(n1)—(CH₂—CH₂—O)_(n2)—(CH₂)— or—((CH₂)_(n1)—(O—CH₂—CH₂)_(n2)—(CH₂)_(n-1)—), where n1 is 1 to 6, 1 to 5,1 to 4, or 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2to 8, or 2 to 5. A further example of an embodiment is one in which X is—(CH₂—CH₂)—, and where Y is —(CH₂—(CH₂—CH₂—O)₃—CH₂—CH₂—CH₂)— or—(CH₂—CH₂—CH₂—(O—CH₂—CH₂)₃—CH₂)—.

The polymer can include one or more spacers or linkers. Examples ofspacers or linkers include linear or branched moieties comprising one ormore repeat units employed in a water-soluble polymer, diamino and ordiacid units, natural or unnatural amino acids or derivatives thereof,as well as aliphatic moieties, including alkyl, aryl, heteroalkyl,heteroaryl, alkoxy, and the like, which can contain, for example, up to18 carbon atoms or even an additional polymer chain.

The polymer moiety, or one or more of the spacers or linkers of thepolymer moiety when present, may include polymer chains or units thatare biostable or biodegradable. For example, polymers with repeatlinkages have varying degrees of stability under physiologicalconditions depending on bond lability. Polymers with such bonds can becategorized by their relative rates of hydrolysis under physiologicalconditions based on known hydrolysis rates of low molecular weightanalogs, e.g., from less stable to more stable, e.g., polyurethanes(—NH—C(O)—O—)>polyorthoesters (—O—C((OR)(R′))—O—)>polyamides(—C(O)—NH—). Similarly, the linkage systems attaching a water-solublepolymer to a target molecule may be biostable or biodegradable, e.g.,from less stable to more stable: carbonate (—O—C(O)—O—)>ester(—C(O)—O—)>urethane (—NH—C(O)—O—)>orthoester (—O—C((OR)(R′))—O—)>amide(—C(O)—NH—). In general, it may be desirable to avoid use of a sulfatedpolysaccharide, depending on the lability of the sulfate group. Inaddition, it may be less desirable to use polycarbonates and polyesters.These bonds are provided by way of example, and are not intended tolimit the types of bonds employable in the polymer chains or linkagesystems of the water-soluble polymers useful in the modified aldehydetagged polypeptides disclosed herein.

Formulations

The conjugates (including antibody conjugates) of the present disclosurecan be formulated in a variety of different ways. In general, where theconjugate is a polypeptide-drug conjugate, the conjugate is formulatedin a manner compatible with the drug conjugated to the polypeptide, thecondition to be treated, and the route of administration to be used.

The conjugate (e.g., polypeptide-drug conjugate) can be provided in anysuitable form, e.g., in the form of a pharmaceutically acceptable salt,and can be formulated for any suitable route of administration, e.g.,oral, topical or parenteral administration. Where the conjugate isprovided as a liquid injectable (such as in those embodiments where theyare administered intravenously or directly into a tissue), the conjugatecan be provided as a ready-to-use dosage form, or as a reconstitutablestorage-stable powder or liquid composed of pharmaceutically acceptablecarriers and excipients.

Methods for formulating conjugates can be adapted from those availablein the art. For example, conjugates can be provided in a pharmaceuticalcomposition comprising a therapeutically effective amount of a conjugateand a pharmaceutically acceptable carrier (e.g., saline). Thepharmaceutical composition may optionally include other additives (e.g.,buffers, stabilizers, preservatives, and the like). In some embodiments,the formulations are suitable for administration to a mammal, such asthose that are suitable for administration to a human.

Methods of Treatment

The polypeptide-drug conjugates of the present disclosure find use intreatment of a condition or disease in a subject that is amenable totreatment by administration of the parent drug (i.e., the drug prior toconjugation to the polypeptide). By “treatment” is meant that at leastan amelioration of the symptoms associated with the condition afflictingthe host is achieved, where amelioration is used in a broad sense torefer to at least a reduction in the magnitude of a parameter, e.g.symptom, associated with the condition being treated. As such, treatmentalso includes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g. terminated, such that the host nolonger suffers from the condition, or at least the symptoms thatcharacterize the condition. Thus treatment includes: (i) prevention,that is, reducing the risk of development of clinical symptoms,including causing the clinical symptoms not to develop, e.g., preventingdisease progression to a harmful state; (ii) inhibition, that is,arresting the development or further development of clinical symptoms,e.g., mitigating or completely inhibiting an active disease; and/or(iii) relief, that is, causing the regression of clinical symptoms.

In the context of cancer, the term “treating” includes any or all of:reducing growth of a solid tumor, inhibiting replication of cancercells, reducing overall tumor burden, and ameliorating one or moresymptoms associated with a cancer.

The subject to be treated can be one that is in need of therapy, wherethe host to be treated is one amenable to treatment using the parentdrug. Accordingly, a variety of subjects may be amenable to treatmentusing the polypeptide-drug conjugates disclosed herein. Generally, suchsubjects are “mammals”, with humans being of interest. Other subjectscan include domestic pets (e.g., dogs and cats), livestock (e.g., cows,pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs,and rats, e.g., as in animal models of disease), as well as non-humanprimates (e.g., chimpanzees, and monkeys).

The amount of polypeptide-drug conjugate administered can be initiallydetermined based on guidance of a dose and/or dosage regimen of theparent drug. In general, the polypeptide-drug conjugates can provide fortargeted delivery and/or enhanced serum half-life of the bound drug,thus providing for at least one of reduced dose or reducedadministrations in a dosage regimen. Thus, the polypeptide-drugconjugates can provide for reduced dose and/or reduced administration ina dosage regimen relative to the parent drug prior to being conjugatedin an polypeptide-drug conjugate of the present disclosure.

Furthermore, as noted above, because the polypeptide-drug conjugates canprovide for controlled stoichiometry of drug delivery, dosages ofpolypeptide-drug conjugates can be calculated based on the number ofdrug molecules provided on a per polypeptide-drug conjugate basis.

In some embodiments, multiple doses of a polypeptide-drug conjugate areadministered. The frequency of administration of a polypeptide-drugconjugate can vary depending on any of a variety of factors, e.g.,severity of the symptoms, condition of the subject, etc. For example, insome embodiments, a polypeptide-drug conjugate is administered once permonth, twice per month, three times per month, every other week (qow),once per week (qw), twice per week (biw), three times per week (tiw),four times per week, five times per week, six times per week, everyother day (qod), daily (qd), twice a day (qid), or three times a day(tid).

Methods of Treating Cancer

The present disclosure provides methods for delivering a cancerchemotherapeutic agent to an individual having a cancer. The methods areuseful for treating a wide variety of cancers, including carcinomas,sarcomas, leukemias, and lymphomas.

Carcinomas that can be treated using a subject method include, but arenot limited to, esophageal carcinoma, hepatocellular carcinoma, basalcell carcinoma (a form of skin cancer), squamous cell carcinoma (varioustissues), bladder carcinoma, including transitional cell carcinoma (amalignant neoplasm of the bladder), bronchogenic carcinoma, coloncarcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma,including small cell carcinoma and non-small cell carcinoma of the lung,adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma,breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renalcell carcinoma, ductal carcinoma in situ or bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcarcinoma, uterine carcinoma, testicular carcinoma, osteogeniccarcinoma, epithelial carcinoma, and nasopharyngeal carcinoma, etc.

Sarcomas that can be treated using a subject method include, but are notlimited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma,rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be treated using a subject method include,but are not limited to, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Leukemias that can be treated using a subject method include, but arenot limited to, a) chronic myeloproliferative syndromes (neoplasticdisorders of multipotential hematopoietic stem cells); b) acutemyelogenous leukemias (neoplastic transformation of a multipotentialhematopoietic stem cell or a hematopoietic cell of restricted lineagepotential; c) chronic lymphocytic leukemias (CLL; clonal proliferationof immunologically immature and functionally incompetent smalllymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia,and hairy cell leukemia; and d) acute lymphoblastic leukemias(characterized by accumulation of lymphoblasts). Lymphomas that can betreated using a subject method include, but are not limited to, B-celllymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin'sB cell lymphoma; and the like.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. By “average” is meant the arithmeticmean. Standard abbreviations may be used, e.g., bp, base pair(s); kb,kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h orhr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt,nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any purificationprotocol known in the art, including chromatography, such as HPLC,preparative thin layer chromatography, flash column chromatography andion exchange chromatography. Any suitable stationary phase can be used,including normal and reversed phases as well as ionic resins. In certainembodiments, the disclosed compounds are purified via silica gel and/oralumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4^(th) edition, Vol. 15/l, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

The subject compounds can be synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods. A varietyof examples of synthetic routes that can be used to synthesize thecompounds disclosed herein are described in the schemes below.

Example 1

Experiments were performed to synthesize a maytansine derivative forattachment to a hydrazinyl-substituted heteroaryl coupling moietyaccording to embodiments of the present disclosure. Reactions wereperformed according to Scheme 1 shown below.

Synthesis of tert-Butyl(S)-1-(9H-fluoren-9-yl)-3,6-dioxo-5-(2-oxo-2-(tritylamino)ethyl)-2,10,13-trioxa-4,7-diazahexadecan-16-oate(Compound 12)

To a dried 20 mL glass scintillation vial containing a dried pea stirbar was added N-(9-fluorenylmethoxycarbonyl)-N-trityl-L-asparagine (11)(597.0 mg, 1.0 mmol), HATU (380.9 mg, 1.0 mmol), and anhydrous DMF (3mL). The clear, colorless solution was stirred at room temperature for15 min. 2-Methyl-2-propanyl 3-[2-(2-aminoethoxy)ethoxy]propanoate (10)(238.2 mg, 1.0 mmol), DIPEA (258.5 mg, 348.4 μL, 2.0 mmol), andanhydrous DMF (3 mL) were combined in a separate, dried 4 mL glassscintillation vial and added dropwise, slowly, to the stirring solution.The reaction was stirred at room temperature for 2 h, added to H₂O (100mL) and 5 M NaCl (25 mL) in a separatory funnel, and extracted with 5×25mL EtOAc. The organic fractions were combined, washed with H₂O (1×25mL), 1.2 M NaHCO₃ (1×25 mL), and 5 M NaCl (1×25 mL), dried over Na₂SO₄,concentrated, adsorbed onto a Biotage KP C18 HS 12 g samplet, andpurified on a Biotage KP C18 HS 60 g cartridge using a gradient of0-100% CH₃CN in H₂O, giving compound 12 as a white, crystalline solid(708.4 mg, 87% yield).

MS (ESI) calcd for C₄₉H₅₄N₃O₈ [M+H]⁺: 812.4 found 812.4.

Synthesis of(S)-5-(2-Amino-2-oxoethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,10,13-trioxa-4,7-diazahexadecan-16-oicacid (Compound 13)

To a 20 mL glass scintillation vial containing a stir bar was added asolution of TFA (6.8 mL, 88.8 mmol) and triisopropylsilane (0.2 mL, 0.98mmol) in 0.2 mL of H₂O. tert-Butyl(S)-1-(9H-fluoren-9-yl)-3,6-dioxo-5-(2-oxo-2-(tritylamino)ethyl)-2,10,13-trioxa-4,7-diazahexadecan-16-oate(12) (708.4 mg, 0.9 mmol) was added in small portions and the reactionwas stirred at room temperature for 4 h. The solution was evaporated,adsorbed onto a Biotage KP C18 HS 12 g samplet, and purified on aBiotage KP C18 HS 60 g cartridge using a gradient of 0-100% CH₃CN inH₂O, giving 385.3 mg (86%) of compound 13 as a white solid.

¹H NMR (DMSO-d6, 400 MHz) δ 7.95-7.86 (m, 2H), 7.83 (t, J=5.6 Hz, 1H),7.75-7.68 (m, 2H), 7.50 (d, J=8.4 Hz, 1H), 7.45-7.39 (m, 2H), 7.37-7.25(m, 3H), 6.90 (s, 1H), 4.35-4.20 (m, 4H), 3.61-3.56 (m, 2H), 3.50-3.42(m, 4H), 3.41-3.37 (m, 2H), 3.24-3.16 (m, 2H), 2.47-2.38 (m, 4H).

MS (ESI) calcd for C₂₆H₃₂N₃O₈ [M+H]⁺: 514.2 found 514.2.

Synthesis of(1⁴S,1⁶S,3²R,3³R,2R,4S,10E,12E,14R)-8⁶-Chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(2S,15S)-15,17-diamino-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13-diazaheptadecanoate(Compound 15)

To a dried 4 mL glass scintillation vial containing a dried stir bar wasadded(S)-5-(2-amino-2-oxoethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,10,13-trioxa-4,7-diazahexadecan-16-oicacid (13) (98.6 mg, 0.2 mmol), HATU (72.9 mg, 0.2 mmol), and anhydrousDMF (1 mL). The clear, colorless solution was stirred at roomtemperature for 15 min. Deacyl maytansine (14) (122.1 mg, 0.2 mmol),DIPEA (72.8 mg, 98.1 μL, 0.6 mmol), and anhydrous DMF (1 mL) werecombined in a separate, dried 4 mL glass scintillation vial and addeddropwise, slowly, to the stirring solution. The reaction was allowed tostir at room temperature for 2 h, adsorbed directly onto a Biotage KPC18 HS 3 g samplet, and purified on a Biotage KP C18 HS 30 g cartridgeusing a gradient of 0-100% CH₃CN in H₂O, giving compound 15 as a whitesolid (149.1 mg, 69% yield).

MS (ESI) calcd for C₄₃H₆₄ClN₆O₁₄ [M+H]⁺: 923.4 found 923.4.

Example 2

Experiments were performed to synthesize a maytansine modified toinclude a hydrazinyl-substituted heteroaryl coupling moiety (e.g., ahydrazinyl-substituted imidazolyl coupling moiety) according toembodiments of the present disclosure. Reactions were performedaccording to Scheme 2 shown below.

Synthesis of 1H-Imidazole-5-carbaldehyde (Compound 21)

A solution of 1.0 g (0.01 mol) of (1H-imidazol-5-yl)methanol (20) in10.0 mL of MeOH was added 8.86 g (0.10 mol) of MnO₂. The mixture wasstirred at room temperature overnight and filtered. The solvent wasevaporated to afford 0.50 g (51%) of compound 21 as a white solid.

¹H NMR (MeOD, 400 MHz) δ 9.78 (s, 1H), 7.91 (s, 1H), 7.89 (s, 1H).

Synthesis of Methyl 3-(5-formyl-1H-imidazol-1-yl)propanoate (Compound23)

To a solution of 0.25 g (0.003 mol) of 1H-imidazole-5-carbaldehyde (21)and 1.12 g (0.013 mol) of methylacrylate (22) in 5.0 mL of DMF was added0.396 g (0.003 mol) of DBU. The reaction mixture was stirred at roomtemperature overnight and concentrated. The product was isolated byflash chromatography on silica gel to afford 0.15 g of compound 23 as anoil.

¹H NMR (CDCl₃, 400 MHz) δ 9.86 (s, 1H), 7.67 (s, 1H), 7.61 (s, 1H), 4.34(t, J=6.4 Hz, 2H), 3.71 (s, 3H), 2.83 (t, J=6.4 Hz, 2H).

MS (ESI) calcd for C₈H₁₁N₂O₃ [M+H]⁺: 183.1 found 183.2.

Synthesis of 3-(5-Formyl-1H-imidazol-1-yl)propanoic acid (Compound 24)

A solution of 0.900 g (0.005 mol) of methyl3-(5-formyl-1H-imidazol-1-yl)propanoate (23) and 0.622 g (0.015 mol) ofLiOH in 20 mL of THF/H₂O (1:1) was stirred at room temperature for 2 h.The reaction mixture was concentrated and the product was isolated bypreparative HPLC to afford 0.800 g (96%) of compound 24 as a colorlessoil.

¹H NMR (DMSO-d₆, 400 MHz) δ 9.87 (s, 1H), 8.74 (s, 1H), 8.42 (s, 1H),4.37 (t, J=6.6 Hz, 2H), 2.92 (t, J=6.6 Hz, 2H).

Synthesis of3-(5-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-imidazol-1-yl)propanoicacid (Compound 26)

To a solution of 0.065 g (0.387 mmol) of3-(5-formyl-1H-imidazol-1-yl)propanoic acid (24) in 2.0 mL of DCE wasadded 0.120 g (0.425 mmol) of (9H-fluoren-9-yl)methyl1,2-dimethylhydrazine-1-carboxylate (25) (see: Eur. J. Med. Chem. 2014,88, 3) and 50 mg of 4 Å molecular sieves. The mixture was stirred atroom temperature for 30 min and 0.098 g of NaBH(OAc)₃ was added. Thereaction mixture was stirred at room temperature for 24 h, then dilutedwith DCM (5 mL) and extracted with H₂O. The organic layer was washedwith 0.1 M HCl, dried over Na₂SO₄, filtered and the solvent was removedin vacuo. The residue was purified on silica gel to afford 0.020 g (12%)of compound 26 as a white solid.

¹H NMR (CDCl₃, 400 MHz) δ 8.83 (bs, 1H), 7.76 (d, J=7.6 Hz, 2H), 7.55(bs, 3H), 7.39 (dd (app. t), J=7.2 Hz, 2H), 7.32 (m, 3H), 4.9-4.0 (m,7H), 3.20 (m, 1H), 2.85-2.50 (m, 7H), 2.04 (m, 1H).

MS (ESI) calcd for C₂₄H₂₇N₄O₄ [M+H]⁺: 435.2 found 435.4.

Synthesis of(1⁴S,1⁶S,3²R,3³R,2R,4S,10E,12E,14R)-8⁶-Chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(2S,15S)-19-(4-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-imidazol-1-yl)-15-(2-amino-2-oxoethyl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecanoate(Compound 27)

To a solution of 21.7 mg (0.049 mmol)3-(5-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-imidazol-1-yl)propanoicacid (26) in 300 μL of DMF was added 45.0 mg (0.049 mmol) ofmaytansinoid 15, 13 μl (0.098 mmol) of 2,4,6-trimethylpyridine, and 18.5mg (0.049 mmol) of HATU. Monitoring by HPLC, the reaction was stirreduntil the starting materials were consumed. The crude reaction mixturewas purified by reversed phase (C18) flash chromatography using agradient of 0-100% MeCN:water with 0.1% formic acid as an additive toafford 50.2 mg of compound 27 in 77% yield.

MS (ESI) calcd for C₆₇H₈₈ClN₁₀O₁₇ [M+H]⁺: 1339.6; found 1339.5.

Synthesis of(1⁴S,1⁶S,3²R,3³R,2R,4S,10E,12E,14R)-8⁶-Chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(2S,15S)-15-(2-amino-2-oxoethyl)-19-(4-((1,2-dimethylhydrazinyl)methyl)-1H-imidazol-1-yl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecanoate(Compound 28)

To a solution of 50.2 mg (0.038 mmol) of compound 27 in 300 μL of DMFwas added 74 μL of piperidine. The reaction mixture was allowed to standfor 30 min and purified by reversed phase (C18) flash chromatographyusing a gradient of 0-100% MeCN:water. The product was collected in testtubes containing 3 mL of 0.1% formic acid in water. The solvent wasremoved in vacuo to afford 30.3 mg (71%) of compound 28 as the formatesalt.

MS (ESI) calcd for C₅₂H₇₈ClN₁₀O₁₅ [M+H]⁺: 1117.5; found 1117.4.

Example 3

Experiments were performed to synthesize a maytansine modified toinclude a hydrazinyl-substituted heteroaryl coupling moiety (e.g., ahydrazinyl-substituted pyrrolyl coupling moiety) according toembodiments of the present disclosure. Reactions were performedaccording to Scheme 3 shown below.

Synthesis of methyl 3-(3-Formyl-1H-pyrrol-1-yl)propanoate (Compound 32)

To a solution of 1H-pyrrole-3-carbaldehyde (30) (500 mg, 5.26 mmol) inCH₃CN (10 mL) was added methyl acrylate (31) (3.8 mL, 42.08 mmol) andDBU (0.8 mL, 5.26 mmol). The resulting mixture was stirred at refluxovernight. The reaction mixture was cooled to room temperature, pouredinto ice-water and extracted with EtOAc. The combined organic layer waswashed with brine, dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by silica gel column chromatography (PE/EA=2:1) togive 800 mg (84%) of compound 32.

¹H NMR (CDCl₃, 400 MHz) δ 9.72 (s, 1H), 7.33 (s, 1H), 7.00 (s, 1H), 6.61(s, 1H), 4.25 (t, J=6.6 Hz, 2H), 3.70 (s, 3H), 2.81 (t, J=6.6, Hz, 2H).

Synthesis of 3-(3-Formyl-1H-pyrrol-1-yl)propanoic acid (Compound 33)

To a solution of methyl 3-(3-formyl-1H-pyrrol-1-yl)propanoate (32) (800mg, 4.42 mmol) in THF/H₂O=1:1 (10 mL) was added LiOH.H₂O (557 mg, 13.26mmol). The resulting mixture was stirred at room temperature for 0.5 h.The pH was then adjusted to ˜3 with 2 N HCl and extracted with EtOAc.The combined organic layer was dried over Na₂SO₄, filtered andconcentrated to give 650 mg (88%) of compound 33 as a yellow solid.

¹H NMR (DMSO-d₆, 400 MHz) δ 12.44 (s, 1H), 9.62 (s, 1H), 7.64 (s, 1H),6.94 (s, 1H), 6.43 (s, 1H), 4.17 (t, J=6.8 Hz, 2H), 2.77 (t, J=6.8 Hz,2H).

MS (ESI) calcd for C₈H₁₀NO₃ [M+H]⁺: 168.1; found 168.1.

Synthesis of3-(3-((2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrol-1-yl)propanoicacid (Compound 35)

To a solution of 3-(3-formyl-1H-pyrrol-1-yl)propanoic acid (33) (650 mg,3.89 mmol) in THF (15 mL) was added (9H-fluoren-9-yl)methyl1,2-dimethylhydrazine-1-carboxylate (34) (see: Eur. J. Med. Chem. 2014,88, 3) (1.7 g, 6.03 mmol) and molecular sieves (1 g). The mixture wasstirred at room temperature for 30 min. Then, NaBH(OAc)₃ (1.65 g, 7.78mmol) was added in several portions at 0° C. The resulting mixture wasstirred at room temperature overnight, then it was concentrated andcrudely purified by silica gel column chromatography (DCM/MeOH=10:1) togive the crude product which was further purified by prep-HPLC to give300 mg (18%) of compound 35.

¹H NMR (DMSO-d₆, 400 MHz) δ 7.90 (d, j=7.2 Hz, 2H), 7.80-7.50 (b, 2H),7.41 (dd(app. t), J=7.4 Hz, 2H), 7.34 (dd(app. t), J=7.2 Hz, 2H), 6.58(bs, 1H), 6.31 (bs, 1H), 5.87-5.70 (m, 1H), 4.60-4.40 (b, 3H), 4.28 (bs,2H), 3.95 (t, J=6.8 Hz, 2H), 3.75 (b, 1H), 3.13 (bs, 1H), 2.66 (s, 3H),2.49 (s, 3H).

MS (ESI) calcd for C₂₅H₂₈N₃O₄ [M+H]⁺: 434.2; found 434.0.

Synthesis of (1S,1⁶S,3²R,3³R,2R,4S,10E,12E,14R)-8⁶-Chloro-1-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(2S,15S)-19-(3-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrol-1-yl)-15-(2-amino-2-oxoethyl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecanoate(Compound 36)

To a solution of 17.7 mg (0.019 mmol) of3-(3-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrol-1-yl)propanoicacid (35) in 200 μL of DMF was added 17.7 mg (0.019 mmol) ofmaytansinoid 15, 5.1 μL (0.038 mmol) of 2,4,6-trimethylpyridine, and 7.3mg (0.019 mmol) of HATU. Monitoring by HPLC, the reaction was stirreduntil the starting materials were consumed, approximately 1 h. The crudereaction mixture was purified by reversed phase (C18) flashchromatography using a gradient of 0-100% MeCN:water with 0.1% formicacid as an additive which afforded 25.7 mg (99%) of compound 36.

MS (ESI) calcd for C₆₈H₈₉ClN₉O₁₇ [M+H]⁺: 1338.6; found 1338.8.

Synthesis of(1⁴S,1⁶S,3²R,3³R,2R,4S,10E,12E,14R)-8⁶-Chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(2S,15S)-15-(2-amino-2-oxoethyl)-19-(3-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrol-1-yl)-2,3-dimethyl-4,14,17-trioxo-7,10-dioxa-3,13,16-triazanonadecanoate(Compound 37)

To a solution of 25.7 mg (0.019 mmol) of compound 36 in 150 μL of DMFwas added 30 μL of piperidine and the reaction mixture was maintainedfor 30 min. The crude reaction was purified by reversed phase (C18)flash chromatography using a 0-100% MeCN:water gradient to afford 17.4mg (82%) of the the title compound.

MS (ESI) calcd for C₅₃H₇₉ClN₉O₁₅ [M+H]⁺: 1116.5; found 1116.4.

Example 4

Experiments were performed to synthesize a maytansine modified toinclude a hydrazinyl-substituted heteroaryl coupling moiety (e.g., ahydrazinyl-substituted furanyl coupling moiety) according to embodimentsof the present disclosure. Reactions were performed according to Scheme4 shown below.

Synthesis of 4-(Hydroxymethyl)furan-3-carbaldehyde (Compound 41)

A solution of furan-3,4-diyldimethanol (40) (0.50 g, 3.9 mmol) in 10 mLof anhydrous DCM was placed in a 50 mL round-bottom flask equipped witha stirring bar. To this solution, a suspension of Dess-Martinperiodinane (0.83 g, 1.96 mmol) in 5 mL of anhydrous DCM was addedslowly over five minutes at room temperature. The resulting yellowsolution was allowed to stir for 1 h at room temperature, then quenchedwith 10 mL of saturated sodium hydrogen sulfite and extracted with EtOAc(2×25 mL). The combined organics were washed with saturated NaHCO₃,followed by water and brine and dried over sodium sulfate. After removalof solvents under vacuum, the residue was purified on silica gel using10-30% EtOAc in hexanes as eluent to afford 0.095 g (39%) of compound 41as a white solid.

¹H NMR (400 MHz, CDCl₃) δ ppm 9.97 (s, 1H), 8.13 (s, 1H), 7.46 (s, 1H),4.64 (s, 2H), 3.70 (br s, 1H).

MS (ESI) calcd for C₆H₇O₃ [M+H]⁺: 127.0 found 127.1.

Synthesis of (9H-Fluoren-9-yl)methyl2-((4-(hydroxymethyl)furan-3-yl)methyl)-1,2-dimethylhydrazine-1-carboxylate(Compound 43)

To an oven-dried 3 mL scintillation vial equipped with a stirring barwas added 4-(hydroxymethyl)furan-3-carbaldehyde (41) (13 mg, 0.1 mmol),(9H-fluoren-9-yl)methyl 1,2-dimethylhydrazine-1-carboxylate (42) (28 mg,0.1 mmol), followed by 2 mL of anhydrous DCE and 50 mg of 4 Å molecularsieves. The resulting mixture was vigorously stirred at room temperaturefor 15 minutes, and treated with NaBH(OAc)₃ (42 mg, 0.2 mmol) in oneportion at room temperature. Stirring continued for 2 hours at roomtemperature until the reaction was judged complete by TLC analysis. Theproduct was isolated by silica gel column chromatography (20-50%EtOAc/hexanes) to afford 34 mg (87%) compound 43 as a colorless oil.

MS (ESI) calcd for C₂₃H₂₅N₂O₄ [M+H]⁺: 393.2 found 393.2.

Synthesis of (9H-Fluoren-9-yl)methyl1,2-dimethyl-2-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)furan-3-yl)methyl)hydrazine-1-carboxylate(Compound 45)

In an oven-dried 3 mL vial, a solution of (9H-fluoren-9-yl)methyl2-((4-(hydroxymethyl)furan-3-yl)methyl)-1,2-dimethylhydrazine-1-carboxylate(43) (12 mg, 30 μmol) in 1 mL of anhydrous DMF was treated with DIPEA(11 μL, 60 μmol), followed by bis(4-nitrophenyl)carbonate (44) (14 mg,45 μmol) in one portion at room temperature. The resulting yellowsolution was briefly vortexed and allowed to stand for 24 hours at roomtemperature. The reaction mixture was diluted with 1 mL of DCM anddirectly purified on silica gel, eluted with 10-20% EtOAc/hexanes togive 8.0 mg (48%) of compound 45 as a clear, colorless oil.

MS (ESI) calcd for C₃₀H₂₈N₃O₈ [M+H]⁺: 558.2 found 558.2.

Synthesis of(1⁴S,1⁶S,3²R,3³R,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(5S,18S)-1-(4-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)furan-3-yl)-5-(2-amino-2-oxoethyl)-17,18-dimethyl-3,6,16-trioxo-2,10,13-trioxa-4,7,17-triazanonadecan-19-oate(Compound 46)

To a 3 mL vial was added maytansinoid 15 (13 mg, 14 μmol), followed by0.5 mL of anhydrous DMF and 5 μL (30 μmol) of DIPEA. To this mixture, asolution of (9H-fluoren-9-yl)methyl1,2-dimethyl-2-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)furan-3-yl)methyl)hydrazine-1-carboxylate(45) (8 mg, 14 μmol) in 0.5 mL of anhydrous DMF was added in one portionat room temperature. The resulting solution was allowed to stand at roomtemperature for 36 hours, then purified by reversed phase chromatography(C18, 10-90% CH₃CN/water) to afford 8.5 mg (44%) of compound 46 as acolorless oil.

MS (ESI) calcd for C₆₇H₈₅ClN₈NaO₁₉ [M+Na]⁺: 1363.6 found 1363.5.

Synthesis of (1S,1⁶S,3²R,3³R,2R,4S,10E,12E,14R)-8⁶-chloro-1-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(5S,18S)-5-(2-amino-2-oxoethyl)-1-(4-((1,2-dimethylhydrazinyl)methyl)furan-3-yl)-17,18-dimethyl-3,6,16-trioxo-2,10,13-trioxa-4,7,17-triazanonadecan-19-oate(Compound 47)

A solution of compound 46 (8 mg, 6 μmol) in 1 mL of anhydrous DMA wastreated with 13 μL (0.12 mmol) of piperidine at room temperature. Thereaction mixture was briefly vortexed and let stand at room temperaturefor 20 minutes until deprotection was judged complete by HPLC analysis.The product was isolated by reversed phase chromatography (C18, 0-50%CH₃CN/water gradient) to afford 4.0 mg (60%) of compound 47 as acolorless film.

MS (ESI) calcd for C₅₂H₇₆ClN₈O₁₇ [M+H]⁺: 1119.5 found 1119.5.

Example 5 General Procedure for Conjugation of a Drug to anAldehyde-Tagged Antibody

To conjugate a hydrazinyl-substituted heteroaryl-modified drug to analdehyde-tagged antibody as described herein, the following generalprotocol was used. 1.0 mM hydrazinyl-substituted heteroaryl-modifieddrug (e.g., hydrazinyl-substituted imidazolyl couplingmoiety-linker-Maytansine) was reacted with 13.5 μM aldehyde-taggedantibody (e.g., an aldehyde-tagged antibody (dimer) with one aldehydetag per chain) in 50 mM sodium citrate, pH 5.5, 50 mM NaCl with 1% DMAat 37° C. for 16-24 hours. After the reaction was complete, unreacteddrug was removed using 3 dilution and concentration steps in a 0.5 mL 10MWCO Amicon spin filter. The sample was analyzed using hydrophobicinteraction chromatography (HIC) to determine the drug to antibody ratioDAR (Tosoh #14947; Mobile Phase A: 25 mM NaPO4, 1.5 M NH4 SO4, pH 7.0;Mobile Phase B: 18.75 mM NaPO4, 25% IPA, pH 7.0).

FIG. 1 shows the HIC trace (min. vs. mAU) of the antibody-drugconjugates produced by conjugating the aldehyde-tagged antibody to themaytansine modified to include a hydrazinyl-substituted imidazolylcoupling moiety, as described in the example above. As shown in FIG. 1,the product included unconjugated (3.673 min.), mono-conjugated (4.833min.), and di-conjugated (5.903 min.) antibody-drug conjugates.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A conjugate comprising at least one modifiedamino acid residue of formula (I):

wherein: R¹ and R² are each independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl; one of R³ and R⁴ is a polypeptide and the other isselected from hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl; R⁵ and R⁶ are eachindependently selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or R⁵ and R⁶ are cyclicallylinked to form a 5 or 6-membered heterocyclyl; Z¹ is selected from CR⁷,N, O and S; Z² is C or N; R⁷ is selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; L isa linker; and W is a drug or a detectable label.
 2. The conjugate ofclaim 1, wherein Z¹ and Z² are each N.
 3. The conjugate of claim 1,wherein Z¹ is CR⁷ and Z² is N.
 4. The conjugate of claim 1, wherein Z¹is O and Z² is C.
 5. The conjugate of claim 1, wherein R³ is thepolypeptide.
 6. The conjugate of claim 1, wherein R⁴ is the polypeptide.7. The conjugate of claim 1, wherein R⁵ and R⁶ are each independentlyselected from alkyl and substituted alkyl.
 8. The conjugate of claim 1,wherein R⁷ is hydrogen.
 9. The conjugate of claim 1, wherein: the linkeris of the formula-(T¹-V¹)_(a)-(T²-V²)_(b)-(T³-V³)_(c)-(T⁴-V⁴)_(d)-(T⁵-V⁵)_(e)—, whereina, b, c, d and e are each independently 0 or 1, where the sum of a, b,c, d and e is 1 to 5; T¹, T², T³, T⁴ and T⁵ are each independentlyselected from (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (EDA)_(w),(PEG), (AA)_(p), —(CR¹³OH)_(h)—, 4-amino-piperidinyl (4AP),para-aminobenzyl (PAB), para-amino-benzyloxy (PABO),meta-amino-benzyloxy (MABO), para-amino-benzyloxycarbonyl (PABC),meta-amino-benzyloxycarbonyl (MABC), an acetal group, a disulfide, ahydrazine, a carbohydrate, a beta-lactam, an ester, (AA)_(p)-MABO,(AA)_(p)-MABC, (AA)_(p)-PABO, (AA)_(p)-PABC, MABO-(AA)_(p),MABC-(AA)_(p), PABO-(AA)_(p), PABC-(AA)_(p), (AA)_(p)-MABO-(AA)_(p),(AA)_(p)-MABC-(AA)_(p), (AA)_(p)-PABO-(AA)_(p), and(AA)_(p)-PABC-(AA)_(p); V¹, V², V³, V⁴ and V⁵ are each independentlyselected from the group consisting of a covalent bond, —CO—, —NR¹¹—,—CONR¹¹—, —NR¹¹CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO₂—,—SO₂NR¹¹—, —NR¹¹SO₂— and —P(O)OH—; each R¹¹ and R¹³ is independentlyselected from hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, amino acyl, alkylamide, substituted alkylamide, sulfonyl,thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl; w is an integer from 1 to20; n is an integer from 1 to 30; p is an integer from 1 to 20; and h isan integer from 1 to
 12. 10. The conjugate of claim 9, wherein: EDA isan ethylene diamine having the structure:

 wherein q is an integer from 1 to 6, r is 0 or 1, and each R¹² isindependently selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl, or wherein two adjacent R¹²groups are cyclically linked to form a piperazinyl ring; PEG is apolyethylene glycol or a substituted polyethylene glycol; AA is an aminoacid residue; and 4AP is

 wherein each R¹⁴ is independently selected from hydrogen, alkyl,substituted alkyl, polyethylene glycol, substituted polyethylene glycol,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,acyl, acyloxy, acyl amino, amino acyl, alkylamide, substitutedalkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 11.The conjugate of claim 10, wherein each R¹¹ is independently selectedfrom hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl.
 12. The conjugate of claim10, wherein each R¹² is independently selected from hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl, or wherein two adjacent R¹² groups arecyclically linked to form a piperazinyl ring.
 13. The conjugate of claim10, wherein each R¹³ is independently selected from hydrogen, alkyl,substituted alkyl, aryl, and substituted aryl.
 14. The conjugate ofclaim 10, wherein each R¹⁴ is independently selected from hydrogen,alkyl, substituted alkyl, polyethylene glycol, substituted polyethyleneglycol, aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocyclyl, and substitutedheterocyclyl.
 15. The conjugate of claim 10, wherein T¹, T², T³, T⁴ andT⁵ and V¹, V², V³, V⁴ and V⁵ are selected from the following table: T¹V¹ T² V² T³ V³ T⁴ V⁴ T⁵ V⁵ (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n)—CO— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹—(C₁-C₁₂)alkyl —CO— — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl—CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— — — — — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) — — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—(PEG)_(n) —NR¹¹— — — — — — — (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹—(PEG)_(n) —NR¹¹— — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— — — — — — —(C₁-C₁₂)alkyl —CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — — — — — — (C₁-C₁₂)alkyl—CONR¹¹— (PEG)_(n) —CO— (EDA)_(w) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w)— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — — — — —(C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO— (CR¹³OH)_(h) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —SO₂— (AA)_(p) — — —(C₁-C₁₂)alkyl —CO— (CR¹³OH)_(h) —CO— — — — — — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹— (PEG)_(n) —CO— — — (C₁-C₁₂)alkyl—CONR¹¹— substituted —NR¹¹— (PEG)_(n) —CO— — — — — (C₁-C₁₂)alkyl(C₁-C₁₂)alkyl —SO2— (C₁C₁₂)alkyl —CO— — — — — — — (C₁-C₁₂)alkyl —CONR¹¹—(C₁-C₁₂)alkyl — (CR¹³OH)_(h) —CONR¹¹— — — — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) —NR¹¹— — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —P(O)OH— (AA)_(p) — — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) —CO— (AA)_(p) — — — — — (C₁-C₁₂)alkyl —CO— (EDA)_(w) —CO—(CR¹³OH)_(h) —CONR¹¹— (C₁-C₁₂)alkyl —CO— (AA)_(p) — (C₁-C₁₂)alkyl—CONR¹¹— (C₁-C₁₂)alkyl —NR¹¹— — —CO— — — — — (C₁-C₁₂)alkyl —CONR¹¹—(C₁-C₁₂)alkyl —NR¹¹— — —CO— (C1-C1₂)alkyl —NR¹¹— — — (C₁-C₁₂)alkyl —CO—(EDA)_(w) —CO— (CR¹³OH)_(h) —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —(C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— — — — — (C₁-C₁₂)alkyl—CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) — — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —CO— MABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— MABC — (AA)_(p) — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— (AA)_(p) - —NR¹¹— (C₁-C₁₂)alkyl —CO— PABC(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO —(C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p) - — — —PABC-(AA)_(p) (C₁-C₁₂)alkyl —CO— (AA)_(p) —NR¹¹— (PEG)_(n) —CO— (AA)_(p)— PABC-(AA)_(p) — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABO— — — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p) — PABO —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABO — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — MABC — — —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) — PABC — (AA)_(p) —(C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO— MABO — — — — — (C₁-C₁₂)alkyl —CO—(AA)_(p) —NR¹¹— (PEG)_(n) —CO— PABO — — — (C₁-C₁₂)alkyl —CO— (AA)_(p)—NR¹¹— (PEG)_(n) —CO— PABC — — — (C₁-C₁₂)alkyl —CONR¹¹— (PEG)_(n) —CO—MABC — (AA)_(p) — — — (C₁-C₁₂))alkyl —CONR¹¹— (PEG)_(n) —CO— (AA)_(p) —PABC —NR¹¹— — — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p)— PABC — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)p —PABC-(AA)_(p) — (C₁-C₁₂)alkyl —CO— 4AP —CO— (C₁-C₁₂)alkyl —CO— (AA)_(p)— —  —.


16. The conjugate of claim 10, wherein the linker is selected from oneof the following structures:

wherein: each f is independently 0 or an integer from 1 to 12; each n isindependently 0 or an integer from 1 to 30; each y is independently 0 oran integer from 1 to 20; each h is independently 0 or an integer from 1to 12; each p is independently 0 or an integer from 1 to 20; each R isindependently selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl,acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide,sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,heterocyclyl, and substituted heterocyclyl; and each R′ is independentlyselected from hydrogen, a sidechain group of an amino acid, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl,carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide,substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy,aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 17.A pharmaceutical composition comprising: a conjugate of claim 1; and apharmaceutically acceptable excipient.